Methods for identifying inducers and inhibitors of proteolytic antibodies, compositions and their uses

ABSTRACT

Covalently reactive antigen analogs are disclosed herein. The antigens of the invention may be used to stimulate production of catalytic antibodies specific for predetermined antigens assocated with particular medical disorders. The antigen analogs may also be used to permanently inactivate endogenously produced catalytic antibodies produced in certain autoimmune diseases as well as in certain lymphoproliferative disorders.

This application is a divisional application of U.S. patent applicationSer. No. 09/046,373, filed Mar. 23, 1998, now U.S. Pat. No. 6,235,714.

FIELD OF THE INVENTION

This invention relates to the fields of immunology, molecular biologyand medicine. More specifically, the invention provides novel methodsand compositions for stimulating the production of novel catalyticantibodies and inhibitors thereof. The invention also provides methodsfor identifying and isolating naturally occurring catalytic antibodiesexpressed from germline genes. Finally, the invention provides methodsfor synthesizing covalently reactive antigenic analogs which stimulatethe production of catalytic antibodies and/or irreversibly inhibit theactivity thereof.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by numerals inbrackets in order to more fully describe the state of the art to whichthis invention pertains. The disclosure of each of these publications isincorporated by reference herein.

The observation that vasoactive intestinal peptide (VIP) is cleaved byantibodies (Abs) from asthma patients provided early evidence that Absmay possess peptidase activity [1,2] This observation has beenreproduced independently by Suzuki et al [3]. Autoantibody catalysis isnot restricted to catalysis of VIP. Autoantibodies in Hashimoto'sthyroiditis catalyze the cleavage of thyroglobulin [4]. Further evidencefor autoantibody catalysis has been provided by reports of DNaseactivity in Abs from lupus patients [5,6] The bias towards catalytic Absynthesis in autoimmune disease is supported by observations that mousestrains with a genetic predisposition to autoimmune disease produceesterase Abs at higher levels when compared to control mouse strains inresponse to immunization with a transition state analog [7].

Like noncatalytic Abs, peptidase Abs are capable of binding Ags withhigh specificity mediated by contacts at residues from the VL and VHdomains. The purified H and L subunits are known to be independentlycapable of binding antigens (Ags), albeit with lower affinity than theparent Ab. X-ray crystallography of Ab-Ag complexes have shown that theVL and VH domains are both involved in binding the Ag [8]. The precisecontribution of the two V domains varies in individual Ab-Ag complexes,but the VH domain may contribute at a somewhat greater level, becauseCDRH3 tends to be longer and more variable in sequence compared toCDRL3.

The initial complexation of a polypeptide Ag by a peptidase Ab isfollowed by cleavage of one or more peptide bonds. Just prior tocleavage, contacts with the catalytic residues of the antibody areestablished with the peptide bond in the transition state. The abilityto hydrolyze peptide bonds appears to reside in the VL domain. Thisconclusion is based on the cleavage of VIP by polyclonal autoantibody Lchains, monoclonal L chains isolated from multiple myeloma patients andtheir recombinant VL domains, and recombinant L chains raised byimmunization with VIP. The H chains of polyclonal and monoclonal Abs toVIP are capable of VIP binding but are devoid of the catalytic activity[9]. The VH domain can nevertheless influence the peptidase activity by“remote control”, because in binding to VIP remote from the cleavagesite, it can influence the conformation of the binding site as shown bythe peptidase activity of F_(v) constructs composed of the catalyticanti-VIP VL domain linked to its VH domain. The anti-VIP VH domainexerted beneficial effects and an irrelevant VH domain exerteddetrimental effects on the catalytic activity, as evaluated by thevalues of VIP binding affinity and catalytic efficiency. The proposedexistence of distinct catalytic and antigen binding subsites incatalytic Abs is consistent with data that Abs generally contain largecombining sites, capable of accommodating 15-22 amino acids ofpolypeptide substrates [8], and that substrate regions distant from thecleavage site are recognized by the Abs. Thus, the VH domain offers ameans to control the specificity of the catalytic site.

Molecular modeling of the L chain suggested that its Asp1, Ser27a andHis93 are appropriately positioned to serve as the catalytic triad [10].The hydrolysis of VIP was reduced by >90% by substitution of Alaresidues for Ser27a, His93 or Asp1 by site-directed mutagenesis [12].The catalytic activity of the wild type protein was inhibitedselectively by diisopropylfluorophosphate (DFP), a serine proteaseinhibitor, but the residual activity of the Ser27a mutant was refractoryto DFP. The K_(m) of the wild type L chain for VIP (130 nM) wasunaffected by mutations at Ser27a, His93 and Asp1. In contrast,mutagenesis at residues forming the extended active site of the L chain(Ser26, H27d/Asp28) produced increases in the K_(m) values (by 10-fold)and increases in turnover (by 10-fold). These results can be explainedas arising from diminished ground state stabilization. The consequentdecrease of ΔG^(t)cat produces an increase in turnover. Thus, two typesof residues participating in catalysis by the L chain have beenidentified. Ser27a and His93 are essential for catalysis but not forinitial high affinity complexation with the ground state of VIP. Ser26and His27d/Asp28 participate in VIP ground state binding and limitturnover indirectly. See FIG. 1.

The VIPase L chain displayed burst kinetics in the early phase of thereaction, suggesting the formation of a covalent acyl-L chainintermediate, as occurs during peptide bond cleavage by serineproteases. The fluorescence intensity was monitored as a function oftime after mixing the L chain with the substrate Pro-Phe-Arg-MCA. Therewas an immediate increase in fluorescence, corresponding to formation ofthe covalent intermediate, followed by a slower increase, correspondingto establishment of the steady rate. The number of active sites wascomputed from the magnitude of the burst by comparison with thefluorescence yield of standard aminomethylcoumarin. The concentration ofcatalytic sites was estimated at 114 nM, representing about 90% of the Lchain concentration estimated by the Bradford method (125 nM).

The catalytic residues (Ser27a, His93, Asp1) in the anti-VIP VL domainare also present in its germline VL domain counterpart (GenBankaccession number of the germline VL gene, Z72384). The anti-VIP VLdomain contains 4 amino acid replacements compared to its germlinesequence. These are His27d:Asp, Thr28e:Ser, Ile34:Asn and Gln96:Trp. Thegermline configuration protein of the anti-VIP L chain was constructedby introducing the required 4 mutations as described previously [12].The purified germline protein expressed catalytic activity as detectedby cleavage of the Pro-Phe-Arg-MCA substrate at about 3.5 fold lowerlevel than the mature L chain (330±23 FU/0.4 μM L chain/20 min;substrate conc. 50 μM). The data suggest that remote effects due to thesomatically mutated residues are not essential for expression of thecatalytic activity.

The present invention provides novel compositions and methods forstimulating production of catalytic antibodies and fragments thereof.Catalytic antibodies with specificity for predetermineddisease-associated antigens provide a valuable therapeutic tool forclinical use. Provided herein are methods for identifying, isolating andrefining naturally occurring catalytic antibodies for the treatment of avariety of medical diseases and disorders, including but not limited toinfectious, autoimmune and neoplastic disease. Such catalytic antibodieswill also have applications in the fields of veterinary medicine,industrial and clinical research and dermatology.

SUMMARY OF THE INVENTION

According to one aspect of the invention, methods and compositions areprovided herein for stimulating catalytic antibody production topredetermined target antigens, including but not limited to thoseinvolved in pathogenic and neoplastic processes. Covalently reactiveantigen analogs (CRAAs) are described which stimulate the production ofcatalytic antibodies with therapeutic value in the treatment of avariety of medical conditions, including autoimmunity disorders,microbial diseases, lymphoproliferative disorders and cancer. Thecatalytic antibodies of the invention may also be used prophylaticallyto prevent certain medical disorders, including but not limited toseptic shock, systemic inflammatory disease and acute respiratorydistress syndrome.

The covalently reactive antigen analogs, (CRAAs) of the presentinvention contain three essential elements and have the followingformula: X1-Y-E-X2. E is an electrophilic reaction center designed toreact covalently with nucleophilic side chains of certain amino acids; Yis a basic residue (Arg or Lys) at the PI position (first amino acid onthe N-terminal side of the reaction center); and X1 and X2 comprisethree to ten flanking amino acids on the N-terminal and C-terminal sideof the reaction center. The resultant CRAA represents a novelcombination of individual structural elements which act in concert to(a) bind chemically reactive serine residues encoded by the germlinegenes for certain serine protease types of catalytic antibodies (as wellas residues such as Thr and Cys that might acquire their chemicalreactivity via somatic sequence diversification of the germline genes);(b) utilize ion pairing and noncovalent forces to bind structures suchas positively charged Asp/Glu residues that are responsible for thebasic residue cleavage specificity of the germline encoded catalyticsites; and (c) bind antibody combining sites at multiple amino acids viaion pairing and noncovalent forces.

In one aspect of the invention, CRAAs are administered to a livingorganism under conditions whereby the CRAAs stimulate production ofspecific catalytic antibodies. The catalytic antibodies are thenpurified. Antibodies so purified are then adminstered to a patient inneed of such treatment in an amount sufficient to inactivate antigensassociated with a predetermined medical disorder.

According to another aspect of the present invention, methods andcompositions are disclosed for administering immunogenic amounts ofCRAAs combined with an immunogenic amount of a conventional transitionstate analog (TSA) to further stimulate catalytic antibody production.

According to another aspect of the present invention, a method isprovided for treating a pathological condition related to the presenceof endogenously expressed catalytic antibodies. Examples of suchabnormal pathological conditions are certain autoimmune disorders aswell as lymphoproliferative disorders. The method comprisesadministering to a patient having such a pathological condition apharmaceutical preparation comprising covalently reactive antigen analogcapable of irreversibly binding the endogenously produced catalyticantibodies, in an amount sufficient to inhibit the activity of theantibodies, thereby alleviating the pathological condition. In thisembodiment, the CRAA contains a minimal B epitope only to minimize theimmunogenicity of the CRAA.

According to another aspect of this invention, a pharmaceuticalpreparation is provided for treating a pathological condition related tothe presence of endogenously produced catalytic antibodies. Thispharmaceutical preparation comprises a CRAA in a biologically compatiblemedium. Endogenously produced catalytic antibodies are irreversiblybound and inactivated upon exposure to the CRAA. The preparation isadministered an amount sufficient to inhibit the activity of thecatalytic antibodies.

In another aspect of the invention, methods for passively immunizing apatient with a catalytic antibody preparation are provided. Catalyticantibodies are infused into the patient which act to inactivate targeteddisease associated antigens. In an alternative embodiment, should thepatient experience unwanted side effects, the activity of the infusedcatalytic antibodies may be irreversibly inactiviated by administeringthe immunizing CRAA to said patient. Again, the immunogenicity of theCRAA in this embodiment would be reduced via the inclusion of aminimally immunogenic B cell epitope. A T cell universal epitope wouldbe omitted in this CRAA.

In yet an alternative embodiment, the catalytic antibodies of theinvention may be coadministered with antisense oligonucleotides to p53.Such combined therapy should prove efficacious in the treatment ofcancer.

In yet another aspect of the invention, active immunization of patientsis achieved by administering the CRAAs of the invention in aCRAA-adjuvant complex to a patient to be immunized. At least 2subsequent booster injections of the CRAA-adjuvant complex at 4 weekintervals will also be adminstered. Following this procedure, thepatient' sera will be assessed for the presence of prophylacticcatalytic antibodies.

The methods and CRAAs of the present invention provide notableadvantages over currently available compounds and methods forstimulating catalytic antibodies specific for predetermined targetantigens. Accordingly, the disclosed compounds and methods of theinvention provide valuable clinical reagents for the treatment ofdisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a free energy diagram for antibody catalysis involvingstabilization of the substrate ground state (ΔG_(S)) and transitionstate (ΔG_(TS)). ΔG⁺ _(uncat) and ΔG⁺ _(cat) correspond to activationenergies for the uncatalyzed and catalyzed reactions, respectively. Kmis a function of the extent of ground state stabilization (ΔG_(S)).Kcat/Km is a function of the extent of transition state stabilizatinrelative to the catalyst-substrate ground state complex. ΔG_(p) is theproduct ground state.

FIG. 2 is a schematic representation of the domain structure of theepidermal growth factor receptor (EGFR) protein. Ligand, ligand-bindingregion found mainly in domain III; TM; transmembrane domain; CYs,cysteine rich domains; and SP, signal peptide.

FIG. 3 is a schematic diagram of the cloning stratagies proposed forpreparing anti-EGFR catalytic antibodies.

FIG. 4 depicts the structure of the CRAA-EGFR peptide.

FIG. 5 is a diagram depicting Fv construction by overlap extension.

FIG. 6 shows a schematic diagram of the immobilization of a serineprotease reactive fluorophosphate transition state analog. (a)triethylamine, CH2C12; (b) water, THF; (c) DAST; (d) glutaric anhydride,pyridine; (e) DCC, DMAP, triethylamine, fluorescein.

FIG. 7 shows a schematic representation of the structure of gp120. V,variable regions; PND, principal neutralizing determinant; arrow,cleavage site targeted by catalytic antibodies generated using themethods of the present invention.

FIG. 8 is a schematic depiction of the DFP reaction with nucleophillicserine residues.

FIG. 9 is a bar graph showing irreversible inhibition of L chainpeptidase activity by diisopropylfluorophosphate ester conjugated tobiotin (top structure). L chain from clone U19 (1 μM) was incubated for30 minutes with the inhibitor. Unbound inhibitors were removed by gelfiltration. Peptidase activity was measured at 20 nM L chain withradiolabeled VIP substrate. Data are expressed as % inhibition relativeto activity of the L chain subjected to gel filtration without inhibitorpretreatment (about 15,000 cpm).

FIG. 10 depicts exemplary immunogen structures contemplated for use inthe present invention. The box shows the structure around the targetedcleavage site (Lys432-Ala433). Flanking residues are indentical in thethree immunogens. Amino acid numbers are those in full-lenghth gp120.

FIG. 11 is an autoradiogram of a non-reducing gel showing the hydrolysisof ¹²⁵I-gp120 (100 nM) incubated with 50 nM IgG from a lupus patient(lane 2, left panel) and 11 nM L chains from MRL/lpr mice (lane 2, rightpanel). Lane 1 in the left and right panel show equivalent amounts ofthe substrate incubated with noncatalytic IgG from an HIV-1 positivesubject and L chains from BALB/c mice. Incubation, 2 hours at 37° C.

FIG. 12 is a graph showing antibody catalyzed cleavage of ¹²⁵I-gp120incubated for 1 hour with lupus IgG (50 nM) without and with DFP (10μM). (B) ¹²⁵I-gp120 from various strains incubated for 2 hours with Lchain Lay2 (1 μM).

FIG. 13 is an immunoblot of a reducing SDS-gel showing hydrolysis ofunlabeled gp120 (11 μM; SF2, Chiron) by L chain Lay2 (20 μM) (Lane 2).

FIG. 14 is a schematic drawing of the putative transition state ofacyl-enzyme formation during peptide bond cleavage by serine proteases.The acyl enzyme complex (right structure) is deacylated by an attackingwater molecule.

FIG. 15 is an exemplary CRAA designed to elicit catalytic antibodies toTNFα.

FIG. 16 is an exemplary CRAA designed to elicit catalytic antibodies toIL-1β.

FIG. 17 is an exemplary CRAA designed to elicit catalytic antibodies toIL1-β. In this CRAA the electrophillic reaction center comprises aboronate molecule.

FIG. 18 is a schematic diagram of the cellular molecules whichparticipate in p53 mediated signalling events.

FIGS. 19A and 19B depict a list of antigens targeted by conventionalmonoclonal antibodies showing clinical promise. Such antigens aresuitable targets for the catalytic antibodies of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Methods are disclosed for stimulating synthesis of catalytic antibodiesof predetermined specificity by the immune system. In one embodiment ofthe invention compositions and methods are provided for the generationof catalytic antibodies to a peptide antigen of choice. In anotherembodiment, compositions and methods are provided which are useful inpassive immunotherapy modalities for the treatment of cancer and othermedical conditions. Catalytic antibodies for treatment of disorders inwhich TNFα and ILβ1 play a key role are also contemplated for use in thepresent invention. Such disorders include, but are not limited to,ischemia and reperfusion injury, septic shock, SIRS, acute respiratorydistress syndrome, rheumatoid arthritis, inflammatory bowel disease,multiple schlerosis and neurotrophic pain.

In another embodiment of the invention, vaccination protocols aredescribed which elicit catalytic Ab production to predetermined viral orpathogenic antigens. The covalently reactive antigen analogs disclosedpreferentially stimulate the production of catalytic antibodies. Suchantibodies provide superior protection against infection due to thepresence of catalytic action against the target antigen which results inits permanent inactivation. Additionally, a single catalytic Ab moleculemay be reused to inactivate multiple antigen molecules as compared tononcatalytic Abs which bind antigen reversibly and stoichiometrically.

Immunization with TSAs [1] has been proposed as a means to derive Absthat can bind the transition state, and thus lower the activation energybarrier for the reaction. The commonly used phosphonate analogs containa tetrahedral phosphorous atom and a negatively charged oxygen atomattached to the phosphorous. Formation of the transition state ofpeptide bond cleavage is thought to involve conversion of the trigonalcarbon atom at the cleavage site to the tetrahedral state, andacquisition of a negative charge by the oxygen of the carbonyl group.The conventional phosphonate TSAs may induce, therefore, the synthesisof Abs capable of binding the oxyanion structure and the tetrahedralconfiguration of the transition state. However, Abs to these TSAs, whilecapable of accelerating comparatively undemanding acyl transferreactions, cannot effectively catalyze peptide bond cleavage. Anantibody to a phosphinate TSA has recently been reported to slowlycleave a stable primary amide [11]. It is possible that theanti-phosphinate Ab may permit superior transfer of a proton to theamide nitrogen at the scissile bond, compared to the more commonanti-phosphonate Abs, which might account for its better catalyticactivity.

Most enzymologists hold that phosphonate TSAs fail to elicit efficientcatalytic Abs because they are poor transition state mimics, and becausemultiple transition states are involved. Enzymes use activated aminoacid sidechains to catalyze peptide bond cleavage. For instance, the Serhydroxyl group acquires enhanced nucelophilicity and the capability tomediate covalent catalysis due to formation of an intramolecular,hydrogen bonded network of the Ser, His and Asp residues. Thephosphonate analogs do not contain structural elements necessary to bindthe nucleophilic reaction center. Induction of the covalent catalysiscapability in Abs is therefore unattainable using conventionalphosphonate TSAs. Further, these TSAs do not exploit the existence ofthe germline encoded, serine protease site in Abs.

Methods are disclosed for the preparation of electrophilic CRAAs whichare capable of reacting with the nucleophilic serine residue of thecatalytic Abs. These novel antigen analogs will be applied to selectcatalysts from the antibody libraries. The logical extension of thisstrategy is to force the utilization of the serine protease sites forthe synthesis of antibodies specific for individual target antigens,such as the EGFR. This can be achieved by immunization with theaforementioned electrophilic CRAAs. Such CRAAs promote clonal selectionof B cells expressing the germline encoded serine protease sites ontheir cell surface. Further, the specificity for EGFR, for example, willbe ensured by incorporating an appropriate antigenic epitope from EGFRwhich will flank the covalently reactive antigen analog structure.

Catalytic Ab synthesis has been documented in autoimmune disease [2, 4].Further, the immune system is capable of producing Abs that catalyze thecleavage of exogenous antigens, including the cleavage of HIV proteingp120. However, patients infected with the virus do not mount acatalytic Ab response to gp120. The HIV CRAAs discussed herein willforce the immune systme to synthesize protective catalytic antibodies toHIV. Data are presented herein which support this approach. gp120 hasbeen selected as the target antigen for the following reasons: (a) It isan essential constituent of HIV-1 for productive infection of hostcells; (b) As a virus-surface protein, gp120 is readily accessible toAbs; and (c) Certain anti-gp120 Abs have been shown to arrest HIVinfection.

The catalyst VL genes can be recruited for the synthesis of HIV-specificcatalytic Abs, by immunization with the CRAAs of the present invention.The analogs are capable of binding the nucleophilic, germline encodedcatalytic site, and therefore, preferentially stimulate the clonalexpansion of B cells producing the catalytic Abs. When necessary,phosphonate TSAs can be combined with CRAAs to induce catalyticantibodies that contain an oxyanion hole in addition to nucleophillicchemical reactivity.

CRAAs reactive with the key structural elements of serine protease-likecatalysts will be synthesized which contain a model B cell epitope ofgp120 involved in CD4 binding (residues 421-436). Autoimmune andnon-autoimmune mice wil be immunized with the B epitope and its CRAAusing procedures well known to those of skill in the art. A T helperepitope will also be incorporated in the CRAA. Individual structuralfeatures known to contribute in serine protease catalysis, i.e., anucleophilic serine residue, an oxyanion hole forming residues, shapecomplementarity with the tetrahedral geometry of the scissile bond, andrecognition of flanking residues in the substrate will be recruited inthe elicited antibodies by incorporating the following features in theTSAs: an electrophilic, tetrahedral phosphonate ester or a negativelycharged phosphonate flanked by the B epitope residues.

The CRAAs of the invention and the resulting catalytic antibodies haveat least three major applications. The first application is directed tothe generation of catalytic antibodies in either humans or animalsfollowing immunization with a CRAA designed for a particular medicaldisorder. The catalytic antibodies so generated would then beadministered to patients to inactivate targeted antigen moieties. Inthis scenario, should the patient experience adverse side effects, theimmunizing CRAA may be administered to irreversibly inactivate thecatalytic antibody. The CRAAs in this embodiment would be synthesizedwith a B cell epitope only in order to minimize immunogenicity.

In the second application, CRAAs may be administered to patients for thepurposes of actively immunizing the patient against particularpathological to generate a state of protective immunity. These CRAAswould be administered as a CRAA-adjuvant complex.

Finally, the CRAAs of the invention may be administered to patients whoare currently expressing catalytic antibodies in association with amedical disorder such as autoimmune disease or multiple myeloma. CRAASmay be designed with specifically react with the antibodies present.Inhibition of catalytic function should result in an amelioration of thedisease state. Again, these CRAAs are designed to contain a minimallyimmunogenic B cell epitope only.

The detailed description set forth below describes preferred methods forpracticing the present invention. Methods for selecting and preparingCRAAs, stimulating the production of catalytic antibodies topredetermined disease antigens are described, as well as methods foradministering the CRAAs or catalytic antibodies in vivo.

I. Selection and Preparation of CRAAs

The covalently reactive antigen analogs of the invention are preparedusing conventional organic synthetic schemes. The novel CRAAs of theinvention contain an electrophilic center flanked by peptide residuesderived from proteins associated with a particular peptide antigen to betargeted for cleavage and the intended use of the CRAA.

Selection of suitable flanking amino acid sequences depends on theparticular peptide antigen targeted for cleavage. For example, viralcoat proteins, certain cytokines, and tumor-associated antigens containmany different epitopes. Many of these have been mapped usingconventional monoclonal Ab-based methods. This knowledge facilitates thedesign of efficacious covalently reactive antigen analogs useful ascatalytic antibody inhibitors as well as inducers of catalyticantibodies with catalytic activities against predetermined targetantigens.

The amino acids flanking the reaction center represent the sequence ofthe targeted epitope in defined polypeptides that play a role indisease, or to which autoantibodies are made in disease.

The structural features of the CRAAs are intended to permit specific andcovalent binding to immature, germline encoded antibodies as well asmature antibodies specialized to recognize the targeted epitope. Basedon the tenets of the clonal selection theory, the CRAAs are alsointended to recruit the germline genes encoding the catalytic antibodiesfor the synthesis of mature antibodies directed towards the targetedepitope.

Polypeptides to be targeted include soluble ligands and the membranebound receptors for these ligands.

Microbial proteins are also intended to targeted for catalysis by theantibodies of the present invention. These include but are not limitedto gp120, gp160, Lex1 repressor, gag, pol, hepatitis B surface antigen,bacterial exotoxins (diptheria toxin, C. tetani toxin, C. botulinumtoxin, pertussis toxin).

Neoplastic antigens will also be incorporated into therapeuticallybeneficial CRAAs. These include but are not limited to EGF, TGFα, p53products, prostate specific antigen, carcinoembryonic antigen,prolactin, human chorionic gonadotropin, c-myc, c-fos, c-jun,p-glycoproteins, multidrug resistance associated proteins,metalloproteinases and angiogenesis factors.

Receptors for neoplastic antigens will also be targeted forantibody-mediated catalysis. These include EGFR, EGFR mutants, HER-2,prolactin receptors, and steroid receptors.

Inflammatory mediators are also suitable targets for catalysis.Exemplary molecules in this group include TNF, IL-1beta, IL-4 as well astheir cognate receptors.

Preexisting catalytic antibodies are found in autoimmune disease andlymphoproliferative disorders. The harmful actions of these catalyticantibodies will be inhibited by administering CRAAs to patients. CRAAsdesigned to be weakly immunogenic will be administered which covalentlyinteract with antibody subunits with specificity for VIP,Arg-vasopressin, thyroglobulin, thyroid peroxidase, IL-1, IL-2,interferons, proteinase-3, glutamate decarboxylase.

For maximum selectivity, the flanking peptide sequences comprise anepitope which is targeted for cleavage. For example, an epitope presentin the epidermal growth factor receptor is incorporated in a CRAA of thepresent invention. In another embodiment of the invention, an epitopepresent in HIV gp120 is incorporated into a CRAA. An explary CRAA forthe treatment of HIV infectin comprises both a B cell epitope and a Tcell epitope to maximize the immunogenicity of the CRAA. Other CRAAsexemplified herein include those suitable for generating catalyticantibodies to TNF and IL-1β.

II. Administration of CRAAs

CRAAs as described herein are generally administered to a patient as apharmaceutical preparation. The term “patient” as used herein refers tohuman or animal subjects.

The pharmaceutical preparation comprising the CRAAs of the invention areconveniently formulated for administration with a acceptable medium suchas water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents or suitablemixtures thereof. The concentration of CRAAs in the chosen medium willdepend on the hydrophobic or hydrophilic nature of the medium, as wellas the other properties of the CRAA. Solubility limits may be easilydetermined by one skilled in the art.

As used herein, “biologically acceptable medium” includes any and allsolvents, dispersion media and the like which may be appropriate for thedesired route of administration of the pharmaceutical preparation, asexemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the CRAA to beadministered, its use in the pharmaceutical preparation is contemplated.

Conventional immunization methods will applied to induce catalytic Absynthesis. Three intraperitoneal and one intravenous injections of theimmunogens (about 100 μg peptide each) will be administered. The finalimmunization will be carried out intravenously. RIBI will be used in theanimal studies. For human use, alum will be employed as the adjuvant.Alum is approved for human use and has previously been shown to provokeAb synthesis to a B-T epitope similar to those proposed in the presentinvention. RIBI is a low toxicity replacement for Freund's CompleteAdjuvant, and reproducibly facilitates good Ab responses to a variety ofAgs. Analysis of two adjuvants is advantageous because the quality andmagnitude of Ab responses to vaccines can be influenced by adjuvants,via effects of the cytokines and TH subpopulations recruited by theadjuvants on B.

CRAAs may be administered parenterally by intravenous injection into theblood stream, or by subcutaneous, intramuscular or intraperitonealinjection. Pharmaceutical preparations for parenteral injection arecommonly known in the art. If parenteral injection is selected as amethod for administering the molecules of the invention, steps must betaken to ensure that sufficient amounts of the molecules reach theirtarget cells to exert a biological effect.

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art.

The pharmaceutical preparation comprising the CRAA may be administeredat appropriate intervals, for example, twice a day until thepathological symptoms are reduced or alleviated, after which the dosagemay be reduced to a maintenance level. The appropriate interval in aparticular case would normally depend on the condition and thepathogenic state sought to be treated in the patient.

III. Administration of Catalytic Antibodies

The catalytic antibodies described herein are generally administered toa patient as a pharmaceutical preparation.

The pharmaceutical preparation comprising the catalytic antibodies ofthe invention are conveniently formulated for administration with aacceptable medium such as water, buffered saline, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol and thelike), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents orsuitable mixtures thereof. The concentration of catalytic antibodies inthe chosen medium will depend on the hydrophobic or hydrophilic natureof the medium, as well as the other properties of the catalyticantibodies. Solubility limits may be easily determined by one skilled inthe art.

As used herein, “biologically acceptable medium” includes any and allsolvents, dispersion media and the like which may be appropriate for thedesired route of administration of the pharmaceutical preparation, asexemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the catalyticantibody to be administered, its use in the pharmaceutical preparationis contemplated.

Conventional passive immunization methods will be employed whenadministering the catalytic antibodies of the invention. In a preferredembodiment, Abs will be infused intravenously into the patient. Fortreatment of certain medical disorders, steps must be taken to ensurethat sufficient amounts of the molecules reach their target cells toexert a biological effect. The lipophilicity of the molecules, or thepharmaceutical preparation in which they are delivered may have to beincreased so that the molecules can arrive at their target locations.Furthermore, the catalytic antibodies of the invention may have to bedelivered in a cell-targeted carrier so that sufficient numbers ofmolecules will reach the target cells. Methods for increasing thelipophilicity and targeting of therapeutic molecules, which includecapsulation of the catalytic antibodies of the invention into antibodystudded liposomes, are known in the art.

The catalytic antibodies that are the subject of the present inventioncan be used as antibody fragments or whole antibodies or they can beincorporated into a recombinant molecule or conjugated to a carrier suchas polyethylene glycol. In addition any such fragments or wholeantibodies can be bound to carriers capable of causing the transfer ofsaid antibodies or fragments across cell membranes as mentioned above.Carriers of this type include but are not limited to those described(Cruikshank et al. in the Journal of Acquired Immune DeficiencySyndromes and Human Retrovirology, March 1997).

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art. For example, thehalf-life of syngeneic IgG in the human is about 20 days. Over thisperiod, 60,480 Ag molecules will be cleaved by one molecule of anantibody with a turnover of 2.1/min (which is the turnover of a humananti-VIP L chain isolated from a phage display library [14]. It can beseen, therefore, that the peptidase antibodies can express considerablymore potent antigen neutralizing activity than stoichiometric,reversibly-binding molecules. Note that the antibody light chainsdiscussed here were selected based on their antigen-binding affinity, aprocedure that favors tight binding to the antigen, but will not selectcatalysts with the best turnover. Antibodies produced by immunizationwith CRAAs and isolated by appropriate selection methods, as disclosedhere, will express considerably greater turnover. Such catalyticantibodies can be used to treat disease at substantially lower doses ofcorresponding noncatalytic antibodies.

The pharmaceutical preparation comprising the catalytic antibodies maybe administered at appropriate intervals, for example, twice a weekuntil the pathological symptoms are reduced or alleviated, after whichthe dosage may be reduced to a maintenance level. The appropriateinterval in a particular case would normally depend on the condition andthe pathogenic state sought to be treated in the patient.

CRAAs will be selected that will generate catalytic antibodies forpassive or active immunotherapy that will fulfill the standard criteriafor acceptable prophylatic or therapeutic agents: (1) Cleavage of thetarget peptide antigen by the catalytic antibody will lead to abeneficial change in a pathological process by either functionallyactivating or functionally inactivating the target peptide antigen; and(2) Administation of said catalytic antibodies or the induction of theirproduction in the body by means of immunization with a CRAA will resultin a favorable therapeutic index such that the clinical benefit gainedoutweighs the morbidity associated with andy side-effects. Discussionsof how such criteria are established for the acceptability ofprophylatic or therapeutic agents are common in the art can can be foundin such texts as Guide to Clinical Trials by Bert Spilker, Raven Press,New York, 1991.

Suitable categories of prophylatic or therapeutic target peptideantigens for the practice of the present invention include but are notlimited to cytokines, growth factors, cytokine and growth factorreceptors, proteins involved in the transduction of stimuli intiated bygrowth factor receptors, clotting factors, integrins, antigen receptors,enzymes, transcriptional regulators particularly those involved incellular program (differentiation, proliferation and programmed celldeath) control, other inducers of these cellular programs, cellularpumps capable of expelling anticancer agents, microbial and viralpeptide antigens.

Conventional monoclonal antibodies that act to inhibit the function ofparticular target molecules are among the most common type oftherapeutic agent under development for clinical use by biotechnologyand pharmaceutical companies. Some of these have shown substantialclincal promise and any exposed peptide target antigens that are part ofthe same molecular functional unit are therefore shown to beparticularly well suited as potential targets for the catalyticantibodies that are the subject of the present invention. The catalyticantibodies comtmplated in the present invention will constitute a majorinprovement over such conventional monoclonals becaule of their abilityto affect many target molecules vs. just one and because of theresulting dramatic decrease in the cost of treatment. The availabilityof peptide bonds within these targeted antigens can be determined bymethods well established in the art including but not limited to ademonstration of cleavage following exposure to proteolytic enzymes andcatalytic light chains capable of cleaving a range of peptide bonds.

A listing of some of the antigens targeted by conventional monoclonalantibodies showing clinical promise and the corresponding medicalindications are shown in FIGS. 19A and 19B.

Thus, it is an object of the present invention to provide a covalentlyreactive antigen analog, and a method of producing it, which is capableof 1) provoking the generation of catalytic antibodies specific to apredetermined antigen of the invention and/or 2) irreversibly inhibitingthe catalytic action of catalytic antibodies associated with autoimmunedisease and certain lymphoproliferative disorders. Further objectsreside in providing processes for preparing antigens and theircorresponding antibodies, and in providing assays and methods of usingthese antibodies as beneficial therapeutic agents.

EXAMPLE IA Catalytic Antibodies for Tumor Immunotherapy

Methods for producing catalytic antibodies (Abs) suitable for treatmentof cancer are described in the present example. Such antibodies offersuperior immunotherapy alternatives for cancer treatment by virtue ofthe catalytic function, as cleavage of the target antigen should resultin its permanent inactivation. Further, a single Ab molecule may bereused to inactivate multiple antigen molecules. In comparison,noncatalytic Abs bind antigen stoichiometrically, and the binding isreversible. Upon dissociation from the complex, the antigen may recoverits biological functions.

The tumor-associated antigen, epidermal growth factor receptor (EGFR),will be utilized for the synthesis of an exemplary antigen suitable forstimulating the production of antibodies with enzymatic activities.Previous work on peptidase antibodies has revealed the following: 1)certain Abs are capable of combining the ability to bind individualpeptide antigens with a peptide bond cleaving activity; 2) the peptidasesite is structurally similar to the active site of non-AB serineproteases. This site is located in the VL domain and is encoded by agermline V domain gene(s); and 3) the synthesis of peptidase Abs occursat increased levels in autoimmune disease.

EGFR serves vital roles in the transduction of signals necessary forcellular differentiation and mitosis. See FIG. 2. Further, signalstransduced via EGFR have been implicated in tumor invasiveness andtransformation. Binding of EGF or TGFα to the EGF receptor stimulatesthe receptor tyrosine kinase and autophosphorylation activities.Receptor activation leads to a cascade of intracellular events whichculminate in increased cell proliferation.

Overexpression of the EGFR gene has been associated with a number ofneoplasms, including adenocarcinoma and squamous cell carcinoma of thelung, breast carcinoma, colon gynecological and bladder carcinoma,glioma, hepatocellular and pancreatic carcinoma and prostate carcinomas.The overexpression in some tumors has been attributed to EGFR geneamplification [15].

EGFR is a suitable tumor antigen, as it is expressed at much higherlevels in tumors compared normal tissues. Consequently, Abs to EGFR aresuitable candidates for anti-tumor reagents. Many monoclonal antibodies(Mabs) to EGFR have been raised to specific epitopes of the receptorwhich do not compete with each other for binding EGFR [e.g., 16].Mendelsohn and coworkers have described mouse MAbs raised using the EGFreceptor protein from human A431 epidermoid carcinoma cells as theimmunogen. MAbs which inhibit the binding of EGF to EGFR also inhibitedthe EGF-stimulated tyrosine protein kinase activity which was assayedusing intact cells or solubilized membranes and an exogenous peptidesubstrate. Further, these MAbs inhibited the proliferation of A431 cellsin tissue culture, whereas those incapable of blocking the binding ofEGF to EGFR were without effect on cell proliferation. Further researchshowed that administration of anti-EGF receptor MAbs can inhibit tumorformation by A431 cells in athymic mice. MAbs of different isotypes havebeen shown to inhibit tumor growth in mice, indicating that constantdomain effector functions are probably not critical in the observedantiproliferative activity. Complete inhibition of tumor growth in vivohas been observed, provided that sufficient Ab amounts wereadministered. The few tumors that persist continue to express EGFR,suggesting that their survival is due to inadequate exposure of thecells to the Abs [17].

Using a breast carcinoma cell line as the immunogen, Modjtahedi et al[16] generated several MAbs against EGFR, some of which blocked thebinding of growth factors by EGFR and inhibited the growth of humansquamous carcinoma cell lines. Several additional EGFR-specific Mabshave been prepared using purified EGF receptors or cells expressing highlevels of EGFR as the immunogens [18]. Phase 1 clinical trial ofanti-EGFR Abs for the treatment of malignant gliomas [19,20], head andneck cancer, and lung cancer are under way.

The data reveal that Abs capable of disrupting EGF binding to EGFR maybe utilized in development of agents effective for immunotherapy of EGFRexpressing tumors. An inverse correlation has been noted between EGFRexpression and the levels of BCL-2, a protein that plays an importantrole in overriding programed cell death (apoptosis). Ligand binding byEGFR under certain conditions has been shown to protect tumor cells fromc-myc induced apoptosis. Glioblastoma cells transfected with a mutantEGFR display decreased apoptosis [21]. In principle, therefore, Abs toEGFR may be capable of inducing apoptosis in tumor cells. If thispossibility is valid, the likelihood of complete tumor regression via anapoptotic pathway following treatment with EGFR Abs will bestrengthened.

Abs capable of cleaving EGFR comprise superior immunotherapeutic agentscompared to their noncatalytic counterparts for the following reasons:(a) Cleavage of EGFR at the appropriate peptide bonds should causepermanent loss of the biological activity, whereas EGFR binding by anoncatalytic Ab can be reversible, and dissociation of the Ab willregenerate the biological functions of the EGFR; and (b) A singlecatalyst molecule can cleave multiple substrate molecules, whereasnoncatalytic Abs can only act stoichiometrically.

Three strategies for the prepararation of catalytic Abs are disclosedherein. The first strategy capitalizes on the availability of cloned Ablight chains with peptidase activity. Previous studies have suggestedthat the nonspecific peptidase activity residing in the VL domain can bedirected by the antigen binding specificity of the VH domain. Hybrid Fvconstructs will be generated composed of an available VL domain linkedto EGFR binding VH domains. Following synthesis and expression insuitable expression systems, the Fv constructs will be assessed forspecific EGFR cleaving activity.

The second cloning strategy is based on the observation that certain Absexpressed in autoimmune disease utilize serine protease catalytic sitesencoded by germline VL genes. Mice with an autoimmune disease backgroundwill be immunized with EGFR expressing cells. Following immunization,catalytic Fv domains will be isolated from a phage display library.Catalysts that combine the germline encoded catalytic activity withsomatically acquired specificity for EGFR will selected by binding tocovalently reactive antigen analogs (CRAAs) reactive with nucleophilicserine residues, followed by binding to the extracellular domain ofEGFR.

The third strategy is based on the hypothesis that the immune system canbe forced to utilize the germline encoded catalytic site for synthesisof Abs to EGFR. Mice will be immunized with an electrophilic CRAA of anEGFR peptide capable of preferentially stimulating catalytic Absynthesis. The Ab catalysts so produced will be assessed for theirinhibitory effects on the tumorigenicity of an EGFR-expressing humancell line in vivo using a variety of methods known to those of skill inthe art, i.e., inhibition of EGF-stimulated EGFR autophosphorylation andinhibition of tumor cell growth.

The compositions and methods disclosed herein facilitate the preparationof specific and catalytically efficient EGFR cleaving antibodiessuitable for cancer immunotherapy. FIG. 3 summarizes the approach to betaken. Previous studies have established the feasibility of isolatingAbs capable of catalyzing the cleavage of certain Ags, i.e., VIP,thyroglobulin and gp120. Information from these studies has been appliedin the present invention resulting in the disclosed strategies forpreparing catalytic Abs to EGFR.

The following materials and methods are provided to facilitate thepractice of the present invention.

Materials and Methods

Immunization: Six MRL/lpr mice will be hyperimmunized with EGFRexpressing cells as previously described. Briefly, about 10⁷ A431 cellswill be recovered by trypsinization of tissue culture flasks,resuspended in PBS and administered in RIBI adjuvant to the miceintraperitoneally. Three booster immunizations using about 5×10⁶ cellswill be carried out at ten day intervals. If high level Ab titers arenot reached, booster injections with the soluble extracellular domain ofthe epidermal growth factor receptor (exEGFR) (25 μg) will beadministered. To drive the immune system to generate catalyticantibodies, six MRL/lpr mice will be hyperimmunized i.p. with theTSA-EGFR conjugated to keyhole limpet hemocyanin (KLH) (50 μg protein)in RIBI according to the above scheme. Blood will be obtained from theretro-orbital plexus at ten day intervals.

Expression and purification of exEGFR: The extracellular domain of EGFR(exEGFR, composed of residues 1-621 of EGFR) will be purified from abaculovirus expression system as previously described [22]. Expressionof the exEGFR is done in Sf9 insect cells, which secrete about 2 mg/mlof the exEGFR into the culture supernatant [22]. Purification by a twostep ion exchange chromatography procedure, permits recovery of theprotein at about 95% homogeneity, as determined by SDS-PAGE [22].

Preparation and purification of EGFR-CRAA peptide: The CRAA is composedof three basic elements: an electrophilic phosphonate ester flanked onthe N terminal side by EGFR residues 294-303 and on the C terminal sideby EGFR residues 304-310. See FIG. 4. The electrophilicity resides onthe phosphorous atom, and is intended to trap nucelophilic serineresidues present in Abs. The basic synthesis scheme for synthesis ofsuch CRAAs has been described [23]. Briefly, a phosphinate containingisostere of lysine (EGFR residue 303) is attached to the appropriateflanking peptide sequence. The isostere will be prepared from thediphenylmethylamine salt of hypophosphorous acid and 6-aminohexanal,followed by removal of the diphenylmethyl group in acid [24]. Therequired flanking peptides are prepared by conventional solid phasepeptide synthesis, except that the peptide corresponding to EGFR residue304-310 contains 2-hydroxy-6-aminohexanoic acid instead of the Nterminal lysine. Side chain protected peptides will be attached to thephosphinate structure by classical solution phase peptide synthesismethods. The phosphinate structure will be converted to the phosphonatephenyl ester by oxidative coupling with phenol. The N terminus of theside chain protected CRAA-EGFR peptide will be coupled to KLH by theglutaraldehyde method. The reaction mixture will be separated by gelfiltration, and residual unconjugated peptide in the lower molecularfractions will be analyzed for inorganic phosphorous after completedigestion with perchloric acid. This will permit estimation of theconjugation efficiency.

exEGFR and EGFR-CRAA ELISA: exEGFR or CRAA-EGFR (100 ng/ml) will becoated on PVC 96 well plates, excess protein binding sites blocked with5% albumin, and binding of appropriately diluted serum Abs to theimmobilized antigens will be measured. The extent of the reaction ismeasured by treatment with goat anti-mouse IgG tagged to peroxidase.Controls include the incubation with preimmune sera and with excesssoluble competitor exEGFR. The procedure for measuring exEGFR andCRAA-EGFR binding by cloned Fv constructs is essentially as above,except that the reaction is visualized by treatment with mouseanti-c-myc Ab (the recombinant proteins contain a 10 residue c-myc tag)and anti-mouse IgG tagged to peroxidase.

Fv preparation: The methods described in previous publications[10,14,25] with certain adaptations will be applied, as summarizedbelow. See also FIG. 5. Construction of an Fv cDNA library will be doneas follows: Total RNA is prepared by standard methods from thesplenocytes of immunized mice while minimizing RNase contamination.Libraries of VL cDNA (residues 1-113) and VH cDNA (residues 1-123) willbe produced from the RNA template using reverse transcriptase andappropriate VL or VH forward primers, which contain, respectively, anSfiI restriction site for cloning into the vector and an antisensesequence encoding a peptide linker. The cDNA is amplified by PCR usingTaq, dNTPs and appropriate primers as shown in FIG. 5. The back primersare based on sequences coding for conserved N-terminal amino acids inthe FRl regions. Limited degeneracy has been introduced in the primersto allow amplification of closely related V-gene families (e.g., Kfamilies 2,3,6). 6 back VL primers, 8 VH back primers, 1 VL forwardprimers and 2 VH forward primers are needed. The forward primers aredesigned to anneal to constant region sequences close to the 3′ end ofthe V domain [26]. The VH and VL back primers contain a NotI site forcloning and a sense sequence encoding the linker. The linker is a14-residue, flexible peptide. SfiI and NotI sites are rare cutters,minimizing loss of library diversity at the restriction digestion step.Following completion of the PCR, the amplified cDNA bands of the correctsize are cut from agarose gels, extracted using Geneclean II (BIO 101)and quantitated by EtBr fluorescence (λem 590 nm, λex 302 nm). The VLand VH cDNA species are linked by overlap extension, i.e., annealing oflinker sense and antisense sequences, and filling in of the two strandswith Taq. Individual cDNA species are purified by agarose gelelectrophoresis and Wizard kits (Promega) prior to performing thelinkage reaction.

Cloning and phage display: The library will then be cloned into thephagemid vector pCANTABhis₆ [27]. The vector contains the followingsequence elements: restriction sites, a signal peptide, a gene3structural peptide, a stop codon (amber) between the insert and gene3, ac-myc peptide tag, poly(his)₆, an IPTG-inducible lac promoter and anampicillin resistance gene. The amber codon permits secretion of solubleV domains or their expression as p3-fusion proteins on the phagesurface, depending on the host strain (HB2151 cells recognize amber as astop and TG1 cells recognize amber as Glu). The cDNA and the vector aredigested sequentially with SfiI and NotI followed by ligation using T4DNA ligase. Host cells are transformed by electroporation, clones areselected in kanamycin and the presence of inserts in individual coloniesis confirmed by PCR using primers located in the vector upstream anddownstream of the insert, yielding EtBr-stained bands at 0.7 kb.Addition of helper phage (VCSM13) permits packaging of phage particlesfrom TG1 cell cultures. The particles in the supernatant of the cultureare precipitated twice with 4% PEG, yielding phage ready for theselection procedures described below.

Selection of EGFR binding Fv: exEGFR will be coated on polystyreneplates at a concentration of about 5 μg/ml in PBS. Following removal ofunbound protein and saturation of nonspecific protein binding sites, theplates are incubated with the phage preparations. Unbound phage will beremoved by extensive washing and bound phage particles will be elutedusing a pH 3.0 buffer.

Catalytic VL domain and hybrid Fv library: The hybrid Fv libraries willbe prepared by linking a VL domain already established to possesscatalytic activity (clone U24 [15]) isolated from an unimmunized mouseto VH domains from the EGFR-binding Fv library. The VL domain cDNA willbe reamplified by PCR as above, except that the forward primer willcontain a NotI site for direct cloning into the vector. The VH domainswill be reamplified from the EGFR binding Fv cDNA library using VHprimers described above, except that the forward primer contains alinker antisense sequence. VL/VH linkage will be as above.

Soluble Fv expression and purification: Phagemid DNA from selectedclones is grown in HB2151 cells. Periplasmic extracts contain 2-10 mg/lof the recombinant protein. Chromatography is on Ni-Sepharose (Qiagen).Unbound proteins are removed with a 0.5 M NaCl buffer. Recombinantantibodies are eluted at pH 5 or with imidazole. A second round of metalaffinity chromatography provides pure recombinant proteins, assessed bySDS-electrophoresis, isoelectric focusing, Mono-Q chromatography andN-terminal amino acid sequencing. Each batch of purified protein isanalyzed by gel filtration (Superose 12 column) and by immunoadsorptionwith immobilized anti-c-myc Ab [14] to confirm that the catalyticactivity belongs to the Ab fragments. Chromatographic procedures areconducted using a gradient FPLC system. Amino acid sequencing is doneusing blots of electrophoresis gels by the Protein Structure CoreFacility at the University of Nebraska Medical Center.

Catalyst selection reagents: Two compounds capable of covalent reactionswith nucleophillic serine residues will be prepared. The first compound,a fluorophosphate (FP) bifunctional reagent, is similar to the serineprotease inhibitor DFP shown in previous studies to inhibit thecatalytic activities of Abs. Because of the poor stability of DFP inwater, its direct attachment to a solid support for phage adsorption isimpractical. A bifunctional reagent containing an FP group conjugated toan affinity tag like biotin will be employed which will permitimmobilization of the conjugate on avidin coated solid phase. The FPester will be reacted with biotin activated with N-hydroxysuccinimide(NHS-LC biotin II, Pierce Chemical Co., 1 in FIG. 6, which alsointroduces a long spacer to minimize steric hindrance effects. Thesynthesis will proceed by esterification of phosphate diester 2 withNHS-LC biotin II (1). Compound 2 will be obtained by phosphorylation of4-triisopropylsilyloxy-2-butanol with dichloroisopropyl phosphatefollowed by hydrolysis and deprotection of the monochlorophosphateintermediate. Conversion to the fluorophosphate 3 will be accomplishedat the final step by treatment with diethylaminosulfuryl trifluoride(DAST). Reagent 3 shall by kept in dioxane or other organic solvent tomitigate possible autoreactivity. An aliquot of the organic solutionwill then be transferred into an aqueous solution containing phage togive an effective concentration of 0.1 to 0.5 mM of 3. In the event thatthe chemical autoreactivity of reagent 3 is too severe for practicalapplication we will consider a fluorescein tag as an alternative tobiotin. Fluorescein contains a phenolic OH and a carboxylic ester whichshould be compatible with the fluorophosphate. Fluorescein will beacylated with the phosphate derivative 4 (obtainable by treatment of 2with glutaric anhydride) to form an amide linkage to its aniline group.Fluorination at phosphorus to obtain 5 will be achieved by treatmentwith DAST. See FIG. 6.

A peptide aldehyde matrix will also be prepared as a means to trapserine protease sites. The commercially available arginal-containingligand antipain (N—[—N-carbonyl-Arg-Val-Arg-al]-Phe) will be activatedwith carbodiimide and linked covalently via the carboxyl group of thePhe residue to the amino residues of AH-Sepharose 4B (Pharmacia). Thesynthesis methods are routine, and have been detailed by Pharmacia.

Catalytic Fv Selection: The Fv phage library will be passed through theimmobilized serine protease trapping reagent described above. Unboundphage will be removed by extensive washing. Elution of bound phage willbe done with 0.1 M glycine-HCl, pH 2.2, which is sufficient to disruptbiotin-avidin and fluorescein-antifluorescein interactions. Elutioncould also be done using 0.1-1M hydroxylamine to dissociate thephosphate-serine linkage. Elution of the peptide aldehyde matrix will bedone with weakly acidic buffer (pH 4.5), which favors breakdown of thehemiacetal adduct. Phage particles recovered from the serine proteasebinding matrix will be amplified by growth in TG1 cells and thensubjected to selection for binding to immobilized exEGFR as describedabove for selection of EGFR binding Fv.

Screening for catalytic activity: Fv fragments will be screened forcleavage of exEGFR, the CRAA-EGFR peptide and a nonspecific peptidasesubstrate, Pro-Phe-Arg-methylcoumarinamide (MCA). A protocol has beendeveloped to rapidly purify large numbers of Ab clones based on theirmetal binding capability. Bacterial supernatants are incubated withNi-Sepharose in 96-well plates fitted with a nitrocellulose filter,unbound material removed by washing with neutral pH buffer, and bound Vdomains eluted into a catch plate using a pH 5 buffer. A MilliporeMultiscreen apparatus permits rapid processing. The eluate isneutralized, and Pro-Phe-Arg-MCA (500 μM), [¹²⁵I]exEGFR or[¹²⁵I]EGFR(tyr, 294-310) (about 30,000 cpm) is added. Hydrolysis of thepeptideMCA substrate is determined using a plate reader (λex 360 nm, λem470 nm; cleavage of the amide bond linking Arg to aminomethylcoumarinproduces increased fluorescence). The peptide-MCA substrate is availablecommercially. The EGFR(tyr, 294-310) is a 19 residue synthetic peptidecorresponding to residues 294-310 of EGFR with a tyrosine residue placedat the N terminus to permit the radiolabeling with ¹²⁵I. Preparation ofthe ¹²⁵IexEGFR and [¹²⁵ I]EGFR(tyr, 294-310) is by the standardchloramine-T method. Removal of free ¹²⁵I is on a disposable gelfiltration column or on a reversed-phase HPLC column, respectively.exEGFR cleavage is determined by nonreducing SDS-electrophoresis (4-15%gels) using a PHAST system (Pharmacia) followed by autoradiography usingKodak XAR film and quantitative scanning of band areas using the programImage. The reaction will be evident as the depletion of the 105 kD bandand appearance of smaller radioactive fragments. Care is taken to onlyquantitate the bands lying within the linear response range of the X rayfilm. Cleavage of [¹²⁵I]EGFR(tyr, 294-310) will be determined bymeasuring the radioactivity rendered soluble in 10% trichloroaceticacid. The TCA precipitation procedure is similar to that describedpreviously to determine VIP cleavage [3]. The method will be validatedby comparison with RP-HPLC on a C-18 column. If difficulties areencountered, electrophoresis on 25% PAGE gels can be carried out todiscriminate between the intact peptide and its fragments, as describedpreviously for VIP [28]. Controls will be eluates from bacteriatransformed with vector without a cDNA insert or cDNA encoding anoncatalytic Fv. Dot-blotting with an anti-c-myc Ab as described in [25]permits quantitation of the recombinant protein.

Screening for inhibition of EGF binding: The selected clones will bescreened for their effect on binding of ¹²⁵I-labeled EGF to A431 cellsin 96 well plates using our previously published methods [15, 18]: Thecells (1×10⁵ cells/well) will be plated in the wells and allowed toadhere to the solid phase, ¹²⁵I-labeled EGF (Amersham) and the Fvsolutions will be added (about 1 nM), the reaction mixture incubated for60 min, the wells washed three times in iced binding buffer, and thewells counted for bound ¹²⁵I-labeled EGFR. Controls will include bindingassays conducted in the absence of Fv, and in the presence of excesscompetitor exEGFR.

Assessment of Catalytic Properties

An immunoblotting cleavage assay will be performed to confirm that thecleavage reaction is not due to artefacts associated with radiolabelingof exEGFR. About 1 μg purified exEGFR is treated with the catalyst foran appropriate length of time followed by SDS-PAGE. The gel is blottedonto nitrocellulose and stained with polyclonal rabbit anti-exEGFRfollowed by anti-rabbit IgG-peroxidase. Depletion of immunostainableintact exEGFR and appearance of immunostainable exEGFR fragments willindicate exEGFR cleavage.

Kinetics: Initial rates for the Ab-catalyzed hydrolysis of radiolabeledexEGFR mixed with increasing amounts of unlabeled exEGFR are computedbased on band intensities seen by SDS-electrophoresis andautoradiography. The velocity of exEGFR cleavage is determined from theintensity of the intact substrate band, and the velocities of individualreactions, from the intensity of each product band. Kinetic constants(K_(m), k_(cat)) will be calculated from the rate data fitted to theMichaelis-Menten equation {v=(V_(max)·[S])/(K_(m)+[S])}. Kinetic studieswill also be conducted using synthetic exEGFR peptides as the substrate,a peptide in which only a single peptide bond is cleaved. The use ofsuch a substrate will eliminate complexities associated with multiplesimultaneous reactions. The kinetics of hydrolysis of such a substratewill be determined as described above, except that reversed-phase HPLCwill be employed to separate the products. Quantitation will be bydetermining the area under the product peaks observed at 214 nm.

Cleavage sites: To identify the peptide bonds cleaved by Abs,electrophoretically pure exEGFR will be incubated with the catalyst fora period sufficient to produce about 100 pmoles of product fragments,the fragments will be separated by polyacrylamide gel electrophoresis,blotted onto a PVDF membrane and the immobilized proteins sequenced byN-terminal Edman's degradation at the UNMC Protein Structure CoreFacility. Controls will include exEGFR incubated with an inactive Fv andexEGFR incubated without Fv. At least 5 N-terminal residues of eachfragment will be identified to permit unambiguous assignment of thecleavage sites. Tryptic mapping and FAB-Mass spectrometry to identifyresultant fragments will be considered if necessary, i.e., if theN-terminus is blocked.

Substrate specificity: Along with exEGFR, cleavage of the followingsubstrate will be tested: (a) ¹²⁵I-lysozyme; (b) ¹²⁵I-thyroglobulin; (c)¹²⁵I-IgG; (d) ¹²⁵I-VIP; and (e) various peptide-MCA conjugates.Protocols for assaying the hydrolysis of these substrates are in place.Purified human thyroglobulin, hen lysozyme (Sigma) and human IgG fromserum are labeled with ¹²⁵I by the chloramine-T method and purified bygel filtration [2, 4]. Following incubation of the radiolabeled proteinswith the Abs, the reaction mixtures will be electrophoresed.Autoradiography will permit products to be visualized as smaller-sizedbands (mass of intact thyroglobulin (monomer), lysozyme and IgG: 330 kD,15 kD and 150 kD, respectively). VIP cleavage is measured as the amountof radioactivity rendered soluble in TCA or by RP-HPLC separations [2].Cleavage of substrates containing MCA linked to charged (Arg, Lys, Asp),uncharged (Leu, Ala) and bulky (Phe) amino acids is measured byfluorimetry.

Isolation of Specific EGFR Cleaving Catalysts from Mice Immunized with aCovalently Reactive Antigen Analog of an EGFR Peptide

As mentioned previously, catalytic Ab synthesis is increased inautoimmune disease. To derive high efficiency catalysts, the immunesystem will be further challenged via the immunization with a covalentlyreactive antigen analog, CRAA, of an EGFR peptide (CRAA-EGFR). Thisantigen analog is designed to increase the recruitment of the germline Vgene encoded site for the synthesis of the EGFR-specific catalytic Abs.Further, the CRAA-EGFR will also select for any serine protease-likecatalytic sites formed by somatic means, i.e., V/D/J rearrangement andsomatic hypermutation.

The key structural features of the CRAA-EGFR are: (a) the tetrahedral,electrophilic phosphorous atom capable of binding nucleophilic serineresidues in catalytic Abs; and (b) the lysine residue on the N-terminalside of the phosphorous atom capable of binding catalytic sitesspecialized for cleavage on the C terminal side of basic residues; and(c) ten and seven amino acids, respectively, on the N and C terminalsides of the CRAA structure, corresponding to the sequence of residues294-310 of EGFR.

The phosphorous atom serves as the analog of the scissile peptide bondcarbon atom linking residues 303 and 304 in EGFR. In the phenylesterconfiguration shown in FIG. 4, the phosphorous atom acquires a partialpositive charge, just as the scissile bond carbon atom carries thepartial positive charge required for its reaction with nucleophilicserine residues. Peptidic O-phenylphosphonates have previously beendescribed to be capable of irreversibly inactivating various serineproteases by forming a covalent bond with the oxygen atom of the activesite serine residue [29]. Sampson and Bartlett [23] have established thechemical synthesis protocol to prepare the phenyl ester at thephosphorous atom, and to attach peptide sequences flanking thephosphonate ester.

It should be noted that the CRAA-EGFR described above is distinct fromprevious phosphonate TSAs applied to raise esterase Abs [13]. Theconventional phosphonate TSAs contain an anionic oxygen attached to thephosphorous, which can bind the oxyanion hole found in the catalysts.The phosphonate TSAs, however, do not react with nucleophilic serineresidues in the catalytic site.

A basic residue is incorporated at the P1 position of the CRAA-EGFR toexploit the existence of the germline encoded, basic residue-specificcatalytic site in Abs. The presence of the basic residue, along with thephosphonate phenylester structure, promotes tight binding to catalyticsite, and thus promotes the ability of the CRAA-EGFR to selectivelystimulate the clonal proliferation of B cells synthesizing the catalyticsites.

EGFR residues 294-310 are incorporated in the CRAA-EGFR to promotesynthesis of Abs with EGFR-specific catalytic activity, as opposed tononspecific catalytic activity. This epitope has been selected becauseit is a component of domain III of EGFR, which is the main contributorof the residues constituting the EGE binding site [30]. See FIG. 4.Further, insertional mutagenesis at the N terminal region of thissequence is described to result in reduced EGF binding. As discussedpreviously, the EGF binding site is composed of non-contiguous residues.Thus, conformational disruptions caused by the intended cleavage atposition 303-304 could also indirectly result in impaired EGFR function.

Fv phage display libraries will be prepared from MRL/lpr micehyperimmunized with the CRAA-EGFR. The presence of high affinity serumAbs capable of binding the CRAA-EGFR will be measured by ELISA toconfirm that the mice mount a vigorous Ab response. Fv librarypreparation and selection will be essentially as described [25] exceptthat selection of phages will be carried out using the immobilizedCRAA-EGFR. Screening for catalytic activity will be done as describedhereinabove. The substrate will be an 18 residue peptide containing atyrosine residue at the N terminus followed by 17 residues correspondingto positions 294-310 of EGFR. The tyrosine residue is located distantfrom the intended cleavage site to minimize interference with Fvrecognition. In addition, screening for exEGFR cleavage will beperformed using the conformational epitope of residues 294-310 aspresented in the functional EGFR protein.

The binding affinity of the catalysts for CRAA-EGFR will be determinedby ELISA. Inhibition of EGFR(294-310) cleavage by increasingconcentrations of the CRAA-EGFR will be determined. The CRAA-EGFR willserve as a competitive alternate substrate, with Ki values close to theKd values estimated from the binding assay.

The product fragments generated by cleavage of EGFR(294-310) and ofexEGFR will be identified, permitting deduction of the cleavage site(s).If the recruitment of the catalytic activity occurs mainly because ofthe phenylphosphonate ester structure in the CRAA-EGFR, both substratesought to be cleaved mainly at the peptide bond linking residues 303 and304 (Lys-Lys bond). As discussed above, antigen specific catalysts canbe synthesized by immunization with ground state antigens. Thus,catalysts capable of cleaving EGFR(294-310) at peptide bonds other the303-304 bond should also be identified. One possible target for cleavageis found at the Arg300-Lys301 bond, as the germline encoded activitypresent in the preimmune repertoire recognizes basic residues.

The methods described above provide a series of high affinity, highturnover catalytic Abs that recognize and cleave EGFR at residues303-304, and induce the loss of the EGF binding activity. Inclusion ofEGFR residues 294-310 in the immunogen is ensures recruitment of highaffinity Abs for EGFR. Inclusion of the phenylphosphonate esterstructure induces clonal selection of Abs with a structurally optimizedserine protease catalytic site. Therefore, catalysts superior to thosegenerated in MRL/lpr mice will be synthesized by implementing theEGFR-CRAA strategy outlined here.

Biodistribution and Anti-Tumor Effects in Vivo

To assess biodistribution and growth effects in vivo, athymic micebearing human tumors have been used as a model to study the tumorlocalization and anti-tumor effects of various drugs, toxins and Abs.

The biodistribution of the six most promising catalytic Fv constructs,along with a noncatalytic Fv construct in tumor bearing mice will becompared. The ability of the ¹²⁵I-radiolabeled Fv constructs to bind andcleave the target antigen will be established in preliminary studies.The tissue-to-blood and tumor-to-blood ratios of the Fv constructs willbe calculated. Imaging studies will be carried to out further evaluatethe tumor specificity of the Fv preparations. The presence of thecatalytic function in the Fv constructs might lead to their increaseddissociation from the surface of tumor cells, because the productfragments of the target antigen will likely bind the catalyst weaklycompared to the intact antigen. This might result in lower tumor:bloodratios for the catalysts compared to the noncatalytic Fv. On the otherhand, if the rate of internalization of the Fv into tumor cells is veryrapid, the catalytic function may not influence the biodistributionpattern of the Fv. Autoradiography of tumor sections will be performedto determine the extent to which the Fv constructs are internalized bythe tumor cells.

Target antigen cleaving catalysts with favorable biodistributionprofiles, along with a non-catalytic Fv and an irrelevant FV, will beevaluated for their ability to inhibit the growth of tumor cells inathymic mice. The time to tumor formation (latent period), the number ofmice developing tumors, and the size of tumors will be noted. Tumorgrowth is determined by the relative rates of cell proliferation andcell death. Apoptosis and necrosis are the distinct processes in celldeath. EGFR is thought to be an important regulator of apoptotic celldeath. It is possible that treatment with the catalytic Fv constructsmay result in complete regression of the tumor, because the cells mightbe freed from negative regulation of apoptosis by EGFR. Cryostatsections of the tumors recovered from the animals will be examined byimmunohistochemical methods for markers of proliferation and apoptosis,i.e., ki-67, bcl2 and bax. ki-67 is a proliferation associated antigenpresent throughout the cell cycle and is a reliable marker forevaluating the growing fraction of a tumor cell population. The bcl-2and bax markers will help assess whether the cells are destined toundergo death via apoptosis.

In summary, the catalytic antibodes of the present invention represent abeneficial therapeutic reagent for the treatment of neoplasticdisorders.

EXAMPLE IB Administration of Catalytic Antibodies and Antisense p53 inCombined Chemotherapy Protocol

When a cell suffers damage to its genome there are mechanisms in placein the cell that will determine if the cell will attempt repair itselfor if it will undergo programmed cell death. In order for proliferatingcells to effectively undertake genomic repair, they must be taken out ofcycle. This is achieved by means of the so-called “cell cyclecheckpoints” which allow proliferating cells time to repair genomicdamage rather than passing it on to daughter cells.

FIG. 18 illustrates the central role of normal (wild-type) p53 ininducing one or the other of these two possible responses of cells togenomic damage. Damage to the genome leads to an increased expression ofp53 which, in turn, sets in motion a variety of other events thatproduce the specific cellular response to this damage.

Based on these relationships, certain agents that inhibit p53 function,such as p53 oligos or p53 catalytic antibodies prepared according to thepresent invention and used incombination with a methods that provide forgetting antibodies or antibody fragments accross the cell membrane, canreasonably be expected to both block programmed cell death and preventthe activation of cell cycle checkpoints depending on which event wouldnaturally occur. Attempts to block either of these cellular responses byusing inhibitors that act upstream or downstream of p53 are problematicbecause of the multiplicity of factors involved, FIG. 18.

These important realizations form the scientific basis for proposedtherapeutic uses of p53 oligos and catalytic antibodies to treat cancer,ischemia-reperfusion injury, and septic shock/SIRS.

Description of the Cellular Mechanisms

During cell division three fundamental processes must be coordinated andany associated errors repaired: (1) the centrosomes must be duplicatedand then segregated; (2) the mitotic spindle must be formed, attached tothe chromosomes, and primed for elongation and sister chromatidseparation at anaphase; and (3) the DNA must be replicated and thechromosomes condensed and then segregated by the mitotic spindle toopposing sides of the cell which shortly will become daughter cells.

A surveillance system is in place that interrupts cell division by meansof checkpoints when it detects damage or potential damage to the genome,including any damage incurred during the natural processes justdescribed. Hartwell and Weinhert operationally defined a cell cyclecheckpoint as follows: When the occurrence of cell cycle event B isdependent upon the completion of a prior cell cycle event A, thatdependence is due to a checkpoint if a loss-of-function mutation can befound that relieves the dependence.

This operational definition has been rigorously demonstrated in studiesof yeast cells where three checkpoints have been described: the DNAdamage, spindle and spindle pole body (centrosome equivalent)checkpoints. The DNA damage checkpoint acts at three different positionsin the cell cycle to arrest proliferation when damage is detected: theG1/S and G2/M transitions, and another that monitors progression throughS. Genetic studies have identified many of the checkpoint components inyeasts but the proteins involved have proven to be functionallypleiotropic, making it difficult to establish simple cause-and-effectrelationships. As pointed out by Paulovich et al. (1997), for example,genes required for the DNA damage checkpoint are also involved in DNArepair, programmed cell death and transcriptional regulation. Results ofyeast studies were subsequently extrapolated to mammalian cells wherehomologous components were found (Hartwell et al. 1994).

Many of the genes necessary for cell cycle arrest at one checkpoint arealso necessary in one or both of the other two. p53, for example, hasbeen shown to play a key role in all three (Cross et al. 1995; Fukasawaet al. 1996; Levine 1997). The critical role of p53 in instigating cellcycle arrest at the G1/S transition in response to DNA damage was firstdemonstrated by Kastan and his colleagues (1991) and has since beenextensively researched. Kastan's group examined the human ML-1myeloblastic leukemia cell line that appears to express wild-type p53(exons 5 through 9 were sequenced and shown to be normal). As is truefor normal cells, treatment of these leukemic cells with nonlethal dosesof γ-irradiation or actinomycin D caused both G1/S and G2/M arrest. InML-1 cells, G1/S arrest was associated with a transient 3- to 5-foldincrease in p53 levels that proceeded cell cycle arrest. Caffeinetreatment was found to block both induction of p53 expression and G1/Scell cycle arrest, suggesting that p53 might play role in G1/S arrest inresponse to DNA damage. In keeping with this hypothesis, cells lackingwild-type p53 did not show a G1/S arrest following γ-irradiation.

In a subsequent study, Kastan's group used solid tumor cell lines tostrengthen their hypothesis (Kuerbitz et al. 1992). Introducingwild-type p53 expression under the control of an inducible promotor in acancer cell line lacking p53 expression allowed cells to undergo a G1/Scell cycle arrest following γ-irradiation. It has additionally beenshown that agents causing DNA strand breaks induce p53 and cycle arrestbut that agents such as anti-metabolites, which are simply incorporatedinto DNA, do not (Nelson & Kastan 1994).

The work of Kastan's group and others have made it clear that amedically important group of agents can cause the production of reactiveoxygen species (ROS) leading to the activation of p53-dependentprocesses by causing DNA strand breaks. These agents include a varietyof anticancer treatments such as ionizing radiation and doxorubicin, aswell as natural mediators including nitric oxide.

The effects of mitotic spindle inhibitors have been studied, includingcertain cancer chemotherapeutic agents, on cells taken from mice havinga p53 genetic knockout. Following treatment, cells became tetraploid oroctaploid as a result of undergoing multiple rounds of DNA synthesiswithout completing chromosome segregation. In contrast, normal mousecells underwent a G2/M cell cycle arrest following treatment. In theabsence of spindle inhibitors, 50% of the cells from p53 knockout mice,but not normal mice, became tetraploid by passage 7. Examination of thetissues of the p53 knockout mice also revealed the presence oftetraploid cells, demonstrating that the results obtained in in vitrostudies with cells from these mice were not a culture artifact. Theseobservations confirm earlier reports that show a correlation betweenloss or inactivation of p53 and tetraploidy or aneuploidy.

Similarly, Fuksaswa et al. (1996) demonstrated that cells from p53knockout mice produce abnormal numbers of centrosomes. This appears toexplain why cultured cells from p53 knockout mice become increasinglyaneuploid in culture when, during the same time period, cells from micewith intact p53 remain diploid. Brown et al. (1994) found that p53copurifies with centrosomes isolated from cultured cells, suggesting apossible direct role for p53 in regulating these organelles.

As shown in FIG. 18, p21 is a key mediator of p53-dependent cell cyclearrest in response to genomic damage. p21 binds to a number of cyclinand cyclin-dependent kinase (cdk) complexes as well as to theproliferating cell nuclear antigen (PCNA). Normal levels of p21 appearto be necessary for the formation of cyclin-cdk complexes which, inturn, are necessary for cell cycle progression (El-Deiry et al. 1993).Increased levels of p21 resulting from p53 activation, however, blockcell cycle progression by interfering with the functions of thesecomplexes and with PCNA. In at least some situations, another gene thatis up-regulated by p53 in response to genomic damage, GADD45, also caninstitute cell cycle arrest at the G1/S transition point (Marhin et al.1997).

Alternatively, genomic damage can lead to a p53-dependent induction ofprogrammed cell death instead of cell cycle arrest and repair. Clarke etal. (1993), for example, have shown that thymocytes taken from miceconstitutively homozygous for a deletion in the p53 gene are resistantto the induction of programmed cell death by γ-irradiation or etoposide,but not by glucocorticoid or calcium. Mice heterozygous for p53 deletionwere also relatively resistant to agents that cause DNA strand breaks,but less so than the homozygots. In contrast, thymocytes from mice withintact p53 underwent programmed cell death in response to all of thesetreatments.

Cancer cells that do not express wild-type p53 are often found toundergo programmed cell death if expression of the protein isexperimentally introduced. This has provided a model system for attemptsto arrive at a mechanistic explanation of how p53 can induce programmedcell death. It must be kept in mind, however, that the use of cell linesthat have eliminated wild-type p53 function and have subsequently hadwild-type p53 constitutively expressed experimentally to create a modelfor analyzing endogenous wild-type p53 functions may result inmisleading conclusions.

Johnson et al. (1996) first demonstrated that ROS can function asdownstream mediators of p53-dependent programmed cell death. Theyproduced high level human wild-type p53 expression in cultured human orrat smooth muscle cells (SMC) using adenoviral vectors carrying humanp53 cDNA under the control of a strong promoter. p53 was expressed inboth cell types at equivalent levels, but only in the human cells wasprogrammed cell death induced. Within eight days of infection,essentially all of the human SMC over-expressing p53 were found to bedead. Kinetic studies documented increased levels of p53 and ROS in theSMC four hours following infection with the p53-carrying virus. Threeunrelated antioxidants were shown to block ROS production but not p53over-expression and to block the induction of programmed cell death. Itwas concluded that increased expression of p53 is sufficient to induceprogrammed cell death in at least some normal cell types, and that ROSare a downstream mediator of this induction.

Vogelstein's group (Polyak et al. 1997) used an adenoviral vector tocause the expression of wild-type p53 in human DLD-1 colon cancer cellsthat had inactive endogenous p53 genes. RNA was purified from thesecells 16 hours after viral infection and 8 hours before evidence ofprogrammed cell death. Analysis was conducted using the SAGE techniquewhich allowed the quantitative evaluation of cellular mRNA populations.Approximately 8,000 transcripts were identified. Of these, 14 weremarkedly (greater than 10-fold) and 26 were significantly more abundantin the cells expressing p53. Thirteen of the 14 most highly inducedgenes were identified and several were found to encode proteins thataffect the redox status of cells.

The group hypothesized that p53 might induce programmed cell death bystimulating the production of ROS. Using a fluorescent probe to measureintracellular ROS levels, the investigators found that ROS productionwas induced following infection with the p53-carrying virus, and thatthe levels of ROS continued to increase as programmed cell deathprogressed. Treatment of DLD-1 cells with the powerful oxidant menadioneor hydrogen peroxide only induced the expression of one of the 14 genes,p21, demonstrating that this group of genes was not induced simply as aresult of ROS expression. Neither were these genes induced as the resultof treating the cells with indomethacin or ceramide, two agents that caninduce programmed cell death in the absence of p53 expression.

Time course experiments suggested a sequence of events during which p53transcriptionally activates redox-controlling genes, causing ROSproduction that results in oxidative damage to mitochondria and, inturn, cell death. Inhibition of each of these steps with specificpharmacologic agents demonstrated a cause-and-effect relationshipbetween sequential events.

These findings suggest the following three-step model for p53-inducedprogrammed cell death in DLD-1 cells: (1) p53 transcriptionallyactivates a specific subset of genes that include oxidoreductases; (2)the induced proteins collectively cause an increase in ROS levels; and(3) ROS damages mitochondria, causing leakage of calcium and othercomponents. These components stimulate members of the ICE-like enzymefamily that are consistently involved in the terminal events ofprogrammed cell death.

There also appears to be some variability among different cell types interms of the genes that are transcriptionally up-regulated by wild-typep53 in response to genomic damage. Two of these are Bax, a member of theBCL-2 family that has been shown to sometimes be involved in thep53-dependent induction of programmed cell death, and GADD45, theproduct of which binds to PCNA and thereby can cause a cell cyclecheckpoint arrest. McCurrach et al. (1997), for example, found that inprimary fibroblasts, Bax is one of the effectors of wild-typep53-dependent programmed cell death induced by chemotherapy. In thisstudy, wild-type p53 was found to transcriptionally activate Bax.Neither Bax nor GADD45, however, were among the genes found to beinduced by wild-type p53 in the previously discussed study by Polyak etal. (1997). The potential importance of Bax in the induction ofprogrammed cell death in response to cellular damage caused bychemotherapy has been demonstrated in work by Strobel et al. (1996).This group transfected an expression vector carrying the Bax cDNA intothe SW626 ovarian cancer cell line that lacks functional p53.Transfectants showed a mean 10-fold increase in Bax expression comparedto control cells. The threshold for the induction of programmed celldeath following chemotherapy treatment was substantially reduced in theBax transfectants when the chemotherapeutic agent was paclitaxel,vincristine or doxorubicin, but not when the agent was carboplatin,etoposide or hydroxyurea.

Additional studies involving cancer cell lines that express wild-typep53 and undergo either proliferation arrest or programmed cell deathfollowing treatment with doxorubicin, show that most of the same 14genes that were highly induced in DLD-1 cells following the introductionof p53, were up-regulated both at lower doses of the drug, which causedcell cycle arrest, and at higher doses, which caused programmed celldeath (Polyak et al. 1997). The authors speculated that the criticalfactor in determining whether a cell undergoes cycle arrest orprogrammed cell death is the ability of that cell to cope with oxidativestress. In other words, cells with a low capacity to handle oxidativestress undergo programmed cell death while more resistant cells undergocycle arrest.

The level of oxidative stress that cells are experiencing has beenpositively correlated with their tendency to undergo p53-dependentprogrammed cell death rather than cell cycle arrest and repair followinggenomic damage. Lotem et al. (1996) studied the effects of oxidativestress and cytokines on these phenomena in myeloid leukemia cells.Antioxidants and certain cytokines exhibited a cooperative protection ofthese cells against programmed cell death induced by cytotoxiccompounds. Increasing oxidative stress with hydrogen peroxide treatment,however, augmented the occurrence of the cell death program andincreased the level of protective cytokine treatment needed to preventprogrammed cell death.

Salicylates are known to inhibit the activation of protein kinases andtranscription factors involved in stress responses. Chernov and Stark(1997) found that salicylate reversibly inhibits wild-type p53 frombinding to DNA and consequently inhibits the ability of p53 to inducep21 transcription and programmed cell death following treatment withtoxorubicin or radiation. If the salicylate is washed out within 60hours of the DNA damage, the inhibited p53-dependent events are able togo on to completion.

One factor that in some circumstances influences whether wild-type p53induces cell cycle arrest or programmed cell death following genomicdamage is c-myc. Saito and Ogawa (1995) studied the rat hepatocellularcarcinoma cell line, FAA-HTC1, that constitutively expresses c-myc anddoes not express p53. c-myc expression in these cells was effectivelysuppressed by an antisense L CHK2HRoligonucleotide. Wild-type p53expression was achieved by transfecting a dexamethasone-inducibleexpression vector carrying wild-type p53 cDNA into these cells. Theresults showed that wild-type p53 can act in the same cells as either aninducer of cell cycle arrest or as an inducer of programmed cell deathdepending on the status of c-myc. Wild-type p53 expression resulted inthe induction of programmed cell death in a portion of the cells, butdid not inhibit the proliferation of surviving cells. If c-mycexpression was inhibited, wild-type p53 expression caused an inhibitionof cell proliferation but did not induce programmed cell death.Unregulated expression of c-myc has also been shown by others to becapable of inducing programmed cell death (Evan et al. 1992; Hoang etal. 1994; Lotem & Sachs 1993).

Additional studies have shown that wild-type p53 may in somecircumstances induce programmed cell death without first causing theexpression of other genes. In these situations, wild-type p53-dependentprogrammed cell death occurs in the presence of actinomycin D orcycloheximide, which block RNA and protein synthesis respectively(Caelles et al. 1994). The introduction into Hela cells of a p53expression vector that lacks the terminal p53 amino acid residuesrequired for p53 binding to DNA, for example, has been shown to producep53-dependent programmed cell death (Haupt et al. 1995). That thisobservation supports the non-involvement of p53 in transcription assumeswithout adequate justification, however, that the only way p53 canaffect transcription is by directly binding to the regulatory elementsof genes themselves. These and similar findings (Sabbatini et al. 1995)have been used to support the argument that p53 can induce programmedcell death without affecting transcription.

It is probable, therefore, that wild-type p53 may induce programmed celldeath by means of transcriptionally activating specific sets of genes,by direct protein-protein interactions or by a combination of thesemethods. The induction of programmed cell death, however, does notnecessarily require the expression of wild-type p53. This is clear fromthe observation that p53-knockout mice develop normally as well as thefact that cells lacking wild-type p53 can be induced to undergoprogrammed cell death (Clarke et al. 1993).

As shown in FIG. 18, the multiple pathways that can initiate programmedcell death converge to utilize a common terminal phase involving theinterleukin 1-beta-converting enzyme (ICE-like) family. This enzymefamily is currently known to contain 11 members and can be divided intothree subfamilies: the ICE, CPP32, and Ich-1 subfamily called caspases(Boldin et al. 1996; Chinnaiyan et al. 1996; Duan et al. 1996a & 1996b;Fernandes-Alnemri et al. 1996; Lin & Benchimol 1995; Lippke et al. 1996;Muzio et al. 1996; Wang et al. 1996). Sabbatini et al. (1997), forexample, specifically studied the role of ICE family enzymes in theoccurrence of p53-dependent programmed cell death. They demonstratedthat a peptide inhibitor of the ICE-like protease CPP32 inhibited thecell death program in baby rat kidney cell lines induced byexperimentally expressing wild-type p53 in these cells.

It also appears that wild-type p53 can potentiate the ability of ICEfamily enzymes to cause programmed cell death (Jung & Yuan 1997). Forexample, inactivating wild-type p53 function in COS-1 cells keeps themfrom undergoing programmed cell death when they are transfected with anexpression vector carrying the cDNA for an ICE-like enzyme, whiletransfecting normal COS-1 cells causes them to undergo the deathprogram. Expression vectors carrying either an ICE-like enzyme or atemperature-sensitive p53 mutant were both transfected into COS-1 cellswith inactive endogenous wild-type p53. At the temperature permissivefor wild-type p53 function, the ability of the ICE-like enzyme to causeprogrammed cell death was significantly augmented. Additionalexperimentation showed that the ability of wild-type p53 to potentiatethe induction of programmed cell death by the enzyme was mediated byBax.

OL(1)p53 for the Treatment of Cancer

All cancer treatments in clinical use kill cancer cells by inducingprogrammed cell death in a dose-dependent manner. In some instancesinduction of this program has been shown to be wild-type p53-dependent.At lower doses, many agents that cause genomic damage effect theinduction of a checkpoint in cells with wild-type p53. The checkpointtemporarily arrests cell proliferation, providing time for the damagedcells to repair.

Recent studies by investigators who have not been involved in thedevelopment of OL(1)p53 provide a rationale for why inhibiting wild-typep53 expression can enhance the killing effect of many anticancertreatments on comparable cancer cells with an intact wild-typep53-dependent cell cycle checkpoint. Evidence shows that when the cellcycle checkpoint fails to engage following therapeutic damage to thegenome, cancer cells continue to replicate their DNA in the absence ofmitosis leading to the induction of programmed cell death. Thetherapeutically important result is that, in the context of a blockedcheckpoint, anticancer treatments become much more effective in killingcancer cells.

In some studies, engagement of the checkpoint was prevented bygenetically knocking out the expression of wild-type p53 or one of itsdownstream effectors, particularly p21. Given the irreversible nature ofthese interruptions, it is clear that wild-type p53 is not required forthe induction of programmed cell death under these circumstances. Inother experiments, methylxanthine derivatives such as pentoxifylline orthe protein kinase C inhibitor UCN-01 (7-hydroxystaurosporine), both ofwhich inhibit G2 checkpoint function, were shown to synergize withagents that interrupt the wild-type p53 pathway in further boosting thesensitivity of cancer cells to anticancer agents.

Consistent with this role of wild-type p53, p53 oligos and OL(1)p53 inparticular can synergistically boost the ability of genome-damagingagents to kill cancer cells. Further, at doses optimum for causing amaximal lethal effect on cancer cells, the combination of OL(1)p53 andan anticancer agent did not kill tested normal cell types.

When phosphorothioate oligos such as OL(1)p53 or natural phosphodiesteroligos bind to cells, they induce the cells to increase their productionof free oxygen radicals by a cyclo-oxygenase-dependent mechanism. Theeffect is much more pronounced in ordinary cell cultures carried out in20% (atmospheric) oxygen than at reduced oxygen tensions. These freeradicals can cause genomic damage, and this phenomenon may explain whyOL(1)p53 kills cancer cells in ordinary tissue culture without thenecessity of adding a compound capable of causing genomic damage, suchas a cancer chemotherapeutic agent, and why OL(1)p53 does not killcancer cells cultured under oxygen levels similar to those found in thebody unless a genomic damaging agent is added. Cancer cells pretreatedwith oligos such as OL(1)p53 can be killed with doses of genome-damagingagents which are not cytotoxic to the cells in the absence of the p53oligo. Presumably this synergy can be even further enhanced by G2inhibitors, such as pentoxifylline or UCN-01, that are more effectivewhen used to treat cancer cells with compromised wild-type p53 functionthan those with intact p53 function.

Since phosphorothioates are DNA analogs, it is possible that cancerchemotherapeutic agents with an affinity for DNA would bind to them.This notion was tested using OL(1)p53, which was shown to tightly bindmitoxantrone but not idarubicin or daunorubicin. The interaction betweenmitoxantrone and the oligo substantially reduced the toxic effects ofthe chemotherapeutic agent on cancer cells that did not expresswild-type p53.

In another study of oligo-drug interactions, bioactive metabolites ofacetaminophen known to react with sulfur groups were shown to bind tophosphorothioate oligos including OL(1)p53. This interaction mayinactivate OL(1)p53 and should be determined prior to further clinicaltesting.

Pharmacology and toxicology studies carried out in several species,including Rhesus monkeys, demonstrate that OL(1)p53 has pharmacokineticproperties favorable for its use as a systemic therapeutic agent andthat the oligo is non-toxic even at dose levels well above the expectedtherapeutic level. Sequencing and cell culture studies suggest thatOL(1)p53 suppresses p53 expression in monkey cells as it does in humancells. The oligo, however, does not have any specific effects on cellsor tissues from lower animals.

A Phase I clinical trial of OL(1)p53 as a single agent was carried outin patients with acute myelogenous leukemia or the myelodysplasticsyndrome. The oligo was given by continuous infusion over 10 days, andresults showed the oligo to be nontoxic over a dose range predicted toyield therapeutic levels. No complete responses were seen.

Malignant cells taken from the patients just prior to the start ofOL(1)p53 administration and at various times during the infusion wereput in culture under 20% oxygen. Compared to peripheral leukemic blastcells from the untreated patients, those taken after the start ofOL(1)p53 infusion died more rapidly as a function of the amount ofOL(1)p53 infused into the patient. Similarly, long-term bone marrowcultures set up from leukemia or myelodysplasia patients demonstrated asubstantially reduced capacity to generate malignant cells as a functionof the amount of OL(1)p53 infused into the donor. This suppressionlasted for many months following treatment without any evidence that theeffect was reversible.

The OL(1)p53 clinical trial results are consistent with laboratory datathat strongly suggest that the oligo must be used in conjunction withgenomic damaging anticancer agents in order to be active in patients.Interpretation of cell culture data using cells from patients in thetrial is also consistent with this hypothesis. When cancer cells wereplaced in culture under 20% oxygen, the oligo induced these cells toproduce ROS which served as the genomic damaging agent.

It follows from the above discussion that blocking p53 expression, witha p53 oligo for example, can result in the prevention of (1) cell cyclearrest, allowing time for an attempt at repair, and (2) p53-dependentprogrammed cell death. Since many cancer therapies cause genomic damage,they can also be expected to cause the induction of wild-type p53 inthose cancer cells that express it. Indeed, x-irradiation, topoisomeraseinhibitors, alkylating agents, anthracyclines, spindle poisons andcertain antimetabolites are all known to produce p53-dependent cellcycle arrest (Cross et al. 1995; Kastan et al. 1991; Linke et al. 1996;Tishler et al. 1995). Inhibition of such induction of wild-type p53should increase the toxicity of these anticancer therapies toproliferating cancer cells by allowing the damaged genome to bereplicated, resulting in the production of dysfunctional cells andinducing programmed cell death as well.

A series of publications that address this issue have come from thelaboratories of collaborating investigators at Johns Hopkins and theUniversity of Pennsylvania (McDonald et al. 1996; Waldman et al. 1996 &1997). Isogenic human colon cancer cell lines were used in thesestudies, differing only in their p21 status. The p21−/− cells wereproduced from the p21+/+ cells using homologous recombination. Normally,the induction of p53 by genomic damage leads to induction of p21 by thep53 acting as a transcription factor in directly binding to the p21gene. Newly synthesized p21 then binds to and blocks the function ofproteins required for cell cycle progression, FIG. 18.

The first of these studies (McDonald et al. 1996) sought to determine ifp21−/− HCT116 human colon cancer cells had DNA repair defects whencompared to p21+/+ HCT116 cells (both HCT116 clones have wild-type p53).The p21-deficient clone was found to be two to three times moresensitive to UV damage than the p21-expressing cells when judged byclonogenic survival assays. Further, p21−/− cancer cells had a two- tothree-fold increased frequency of spontaneously arising 6-TG-resistantcolonies indicative of hprt gene inactivation by mutation compared tocells with intact p21 function. These data suggest that the loss of p21function is associated with reduction in the ability of cells to repairDNA damage.

To further test this concept, investigators transfected an expressionvector into p21+/+ or −/− HCT116 human colon cancer cells. The vectorconsisted of a beta-galactosidase cDNA driven by a cytomegalovirusreporter, and was purposely damaged prior to transfection using eitherUV irradiation or a cis-platinum anticancer agent. HCT116 cells lackingp21 were found to be three- to five-fold less efficient at repairing thedamaged expression vector compared to p21+/+ HCT116 cells. Transfectionof an expression vector carrying p21 cDNA into the p21−/− HCT116 cellsincreased their repair capacity two- to three-fold. It was concludedthat agents which inhibit p21 interaction with PCNA, and thus preventcell cycle arrest in response to DNA damage, may have synergisticcytotoxic interactions with classical anticancer agents that cause DNAdamage.

In a subsequent study, investigators examined the effects of certaingenomic damaging agents on p21+/+ and −/− HCT116 cells (Waldman et al.1996). Agents included the cancer therapeutics doxorubicin, etoposideand γ-irradiation as well as the topoisomerase-1 inhibitor camptothecan.Each was shown to be capable of completely killing cultures of thep21−/− cells within 90 hours of treatment by inducing programmed celldeath at concentrations causing p21+/+ cells to undergo a prolonged cellcycle arrest but not cell death. Analysis of the p21−/− cells showedthat, following treatment, the cells were briefly blocked in G2 but notG1 and then began multiple rounds of DNA synthesis in the absence ofmitosis, and that the resulting hyperdiploid cells with abnormal nuclearmorphology subsequently underwent programmed cell death.

A similar set of experiments was conducted using the DLD-1 human coloncancer cell line which, unlike the HCT116 line, has mutated p53 butresembles the HCT116 line in being diploid. The authors reasoned thatp53 mutant cells would not express p21 following DNA damage and would befunctionally equivalent to p21−/− cells. As predicted, DLD-1 cellsexpressed little p21 after treatment with doxorubicin or γ-irradiation,and demonstrated a checkpoint defect that resulted in the occurrence ofessentially the same set of morphologic/physiologic changes as in theHCT116 line that terminate in programmed cell death.

When these experiments were conducted using aneuploid human colon cancercell lines with mutant p53, the effects of treating two of these lineswith DNA-damaging agents were found to follow the same pattern of eventsthat lead to programmed cell death. In the third line, pre-existinganeuploidy was sufficiently pronounced to inhibit any firm conclusionsabout a significant increase in DNA content prior to cell death. Waldmanet al. concluded that “detailed analyses demonstrated that theprogrammed cell death was apparently induced by an uncoupling betweenmitosis and S phase after DNA damage. Instead of undergoing coherentarrest, cells without the p21-dependent checkpoint continued to undergorounds of DNA synthesis in the absence of mitosis, culminating inpolyploidy and programmed cell death” (p. 1034).

However, the authors failed to comment on an important pointdemonstrated by their experiments. Several publications have indicatedthat in cells with wild-type p53, programmed cell death induced by manyanticancer therapeutics is p53-dependent (Dronehower et al. 1992; Loweet al. 1993; Symonds et al. 1994). Further, some cancer cells withwild-type p53 can be more sensitive to chemotherapy than similar cellswith mutated p53 (Aas et al. 1996; Lowe et al. 1993a & 1993b). Yet thefinding that three different colon cancer cell lines with mutated p53underwent a similar series of events leading to the induction ofprogrammed cell death, as in the HCT116 p21−/− cells, suggests thatprogrammed cell death as a result of replicating damaged DNA in theabsence of mitosis is wild-type p53-independent.

The HCT116 studies demonstrate that interruption of p53/p21-dependentcell cycle arrest can lead to a lowering of the threshold for programmedcell death induction by anticancer treatments because programmed celldeath is induced in p21−/− cells at lower doses than is required forHCR116 cells that are p21+/+. Since both the p21+/+ and p21−/− HCT116cells express wild-type p53, this induction could be p53-dependent. Ifbased on a p53-dependent programmed cell death mechanism, the thresholdlevel at which DNA or genomic damage induces programmed cell death mightbe lower in cancer cells that express wild-type p53. In the absence ofwild-type p53, however, there could be a higher damage level thresholdfor the induction of p53-independent programmed cell death.

Because OL(1)p53 transiently inhibits the expression of p53, treatingcancer cells with this oligo plus conventional therapy can cause both aninterruption of cell cycle checkpoints during the time p53 issuppressed, and p53-dependent programmed cell death following therecovery of p53 expression. OL(1)p53, therefore, should be moreeffective at sensitizing cancer cells expressing wild-type p53 toanticancer therapies than the approaches just described involving p53 orp21 genetic knockouts.

Experiments presented in the third monograph of this series weredesigned to determine if inhibition of cell cycle checkpoints wouldincrease γ-irradiation sensitivity of HCT116 human colon cancer cellsgrown in immunocompromised animals (Waldman et al. 1997). Xenografttumors were established from p21+/+ and p21−/− subclones of the cellline. In the absence of treatment, p21+/+ and p21−/− tumors grew atalmost identical rates. Twelve to 17 animals per group with tumors ofapproximately 50 mm² were then treated with either 7.5 or 15 Gy of localγ-irradiation and subsequently measured biweekly. Radiation of animalswith the p21+/+ tumors resulted in no cures, and all of the p21+/+tumors continued to grow for several days following treatment. Incontrast, 18% and 38% of the p21−/− tumors (P<0.01 by chi-square test)were cured by the γ-irradiation as a function of dose where a cure wasdefined as the absence of detectable tumor. p21−/− tumors that were notcured showed substantial dose-dependent decreases in size followingtreatment.

A second objective of this study was to determine the value ofclonogenic survival assays in evaluating cancer therapies influenced bythe p53 and/or p21 status of target cells. It was found that the numberof clones surviving γ-irradiation were few in number, but nearlyequivalent when the p21+/+ and p21−/− subclones of HCT116 were compared.The low colony number was attributed to cell cycle arrest and programmedcell death respectively. In the case of p21+/+ cells, but not p21−/−,the area between surviving colonies consisted of a lawn of viable cells.Investigators pointed out that this lawn of viable p21+/+ cellsfunctioned like a feeder cell layer such as is known to be important insupporting the growth of clonogenic cells. They further argued that theexistence of this feeder layer in the treated p21+/+ tumors and the lackof such a feeder cell population in vivo could explain their animaldata.

Another group also examined the effects of wild-type p53 and/or p21disruption on the sensitivity of cancer cells to certain cancerchemotherapeutic agents and ionizing radiation (Fan et al. 1995 & 1997).MCF-7 human breast cancer cells or HCT116 colon cancer cells, both ofwhich had wild-type p53, were either transfected with a human papillomavirus type-16 E6 gene (MCF-7/E6 or HCT116/E6) or a dominant p53 mutant(MCF-7/mu-p53) to interrupt the wild-type p53 function. Using aclonogenic survival assay, all three subclones with inhibited wild-typep53 function, as well as the HCT116 p21−/− cells, were shown to besignificantly more sensitive to cisplatin and nitrogen mustard than thecorresponding cells with intact wild-type p53 or p21 function.

All four of the subclones with disrupted wild-type p53 or p21 functionwere found to be deficient in their ability to repair transfectedcisplatin-damaged CAT-reporter genes when compared to the correspondingcells with intact wild-type p53 or p21 function. Consequently, theinvestigators attributed the increased cisplatin sensitivity of thesecells to defects in G1 checkpoint control, nucleotide excision repair,or both.

Like the Johns Hopkins group, Fan's group did not see a significantdifference in the clonogenic survival assay between cells with intactwild-type p53 or p21 function and those without it when ionizingradiation was used as the genomic damaging agent. They apparently werenot aware, however, of the shortcomings of this assay as illuminated bythe Johns Hopkins research team.

A survey of the p53 status and radiosensitivity of twenty humansquamous-cell carcinoma cell lines taken from patients with head andneck cancers was conducted by Servomaa et al. (1996). p53 mutationsand/or deletions were found in 15 of the lines. The “mean inactivationdose” (AUC) was determined using a clonogenic survival assay scored fourweeks after radiation treatment. The results were 1.82±0.24 Gy for thelines with mutated or absent p53 and 2.23±0.15 Gy for the lines withwild-type p53 (P<0.01). The authors concluded that the lines with no p53expression were the most radiosensitive.

The methylxanthine derivative pentoxifylline has been found to be a G2checkpoint inhibitor (Russell et al. 1996). It is a relatively nontoxiccompound given to patients with a variety of disorders because of someof its other properties, which include the ability to increase red bloodcell flexibility (Ciocon et al. 1997). Pentoxifylline exhibitedsynergism with cisplatin in killing cancer cell lines with interruptedwild-type p53 function or p21 deficiency without altering thesensitivity of control cells with intact wild-type p53 and p21 (Pan etal. 1995). The drug was also found to be much more effective atinhibiting the G2 checkpoint in cells that had compromised wild-type p53function.

Russell et al. (1996) inactivated p53 function in the human A549 lungadenocarcinoma cell line by transducing the E6 gene from HPV type 16.Using a clonogenic survival assay, they found that both pentoxifyllineand a novel methylxanthine, lisofylline, caused a 15-fold sensitizationof the E6 transduced cancer cells to γ-irradiation when compared tocontrols. Both agents were shown to block the ability of radiation toinduce G2 cell cycle arrest, and lisofylline was found to block G1arrest as well.

UCN-01 (7-hydroxystaurosporine) is a protein kinase C inhibitor that canalso block the G2 checkpoint. It has shown anti-neoplastic activityagainst human tumors grown in rodents and is currently in clinical trailfor cancer treatment (Pollack et al. 1996). Wang et al. (1996) testedthe ability of this agent to influence the. sensitivity to cisplatin ofMCF-7 breast cancer cells with wild-type p53 or p53 inactivated bytransfection of an expression vector carrying the HPV E6 gene. Drugsensitivity was measured using both clonogenic survival and MTT assaysand was shown to be markedly enhanced by UCN-01 treatment in cellslacking intact wild-type p53 function when compared to cells withfunctional wild-type p53. As for the studies involving pentoxifylline,UCN-01 was found to be much more effective in blocking G2 arrest inducedby genomic damage in cells where wild-type p53 function had beeneliminated than in those with intact function.

Similarly, Shao et al. (1997) demonstrated that UCN-01 is much moreeffective in boosting the cytotoxic effects of genomic damaging agentson HCT116 colon and MCF-7 breast cancer cell lines lacking wild-type p53function as a result of experimental manipulation compared to the samecells where this function is intact.

Caffeine also blocks G2 cell cycle arrest in vitro, and appears tooperate by activating p34cdc2 kinase. Yao (1996a) demonstrated thatcaffeine treatment selectively sensitizes tumor cells deficient inwild-type p53 function to radiation-induced programmed cell death. Thusit appears that for some cancers, the use of OL(1)p53 plus a G2checkpoint inhibitor might boost the beneficial effects of conventionalanticancer therapy to a greater degree than OL(1)p53 alone.

Microtubule active agents induce a cell cycle checkpoint that typicallycauses a G2 arrest. Several studies have implicated wild-type p53 asplaying a role in influencing the response of cells to G2 active agents(Fan et al. 1995; Powell et al. 1995; Russell et al. 1995). Thesefindings led Tishler et al. (1995) to examine the ability of the cancerchemotherapeutic agents taxol, vinblastine and nocodazole to inducewild-type p53-dependent processes in the pre-malignant embryonic mouseNIH-3T3 cell line. All three microtubule active agents caused G2 cellcycle arrest and increased p53-DNA binding. Only vinblastine andnocodazole were shown to cause an increase in p21 transcription.

Wahl et al. (1996) extended these studies by looking at the effects ofinterrupting wild-type p53 function on the sensitivity of fibroblasts totaxol. Wild-type p53 function was disrupted in normal human fibroblastsby transfecting them with expression vectors carrying either the HPV E6gene or the SV40 T antigen gene. Fibroblasts also were taken from normaland p53 knockout mice. Compromised p53 function in cells from eithertype correlated with a seven- to nine-fold increase in taxolcytotoxicity compared to controls. Taxol was shown to kill cells byinducing programmed cell death independently of their p53 status. Cellswith intact p53 that survived taxol treatment showed increased levels ofp21 and underwent cell cycle arrest.

In response to genomic damage, wild-type p53 induces, in addition top21, a second gene GADD45 that also functions to induce a cell cyclecheckpoint by means of its inhibiting effect on PCNA. Smith et al.(1996) blocked GADD45 expression in the RKO human colon cancer cell linethat expresses wild-type p53 by transfecting it with an antisensevector. Reducing GADD45 levels sensitized the cancer cells to thekilling effects of UV irradiation and to cisplatin treatment. Inaddition, cells in which GADD45 was suppressed showed a reduced capacityto repair DNA damage as judged by the use of UV-damaged reporterplasmids and unscheduled DNA synthesis experiments. Expression vectorscarrying a variety of genes that disrupt wild-type p53 function werealso transfected into the RKO cells. Suppressing p53 function had thesame effect on DNA repair as suppressing GADD45 expression.

The existence of at least one additional p53-regulated gene, GADD45,that can produce generally the same inductive effects as p21 cell cyclecheckpoints makes p53 a better target than p21 for blocking checkpointinduction by genomic damaging agents.

According to the present invention, inhibitors of EGFR or HER2 such asconventional monoclonal antibodies or preferably catalytic antibodiesgenerated according to the present invention used in combination withp53 oligos such as OL(1)p53 and/or other cell cycle checkpointinhibitors such as UCN-01, p21 oligos or p27 oligos will be particularlywell suited for use incombination with conventional chemotherapy for thetreatment of carcinomas that express EGFR and/or HER2.

Mendelsohn and his coworkers have shown that blocking EGFR function, forexample with a monoclonal antibody, causes an increased expression ofp53 and p21 or p27/KIP1 resulting in the induction of a cell cyclecheckpoint (Wu et al. 1996; Peng et al., 1996). The combined use of aEGFR inhibitor with a p53, p21, or p27 inhibitor such as anoligonucleotide or catalytic antibody will prevent the cell cycle arrestand boost the anticancer effect of the EGFR inhibitor particularly whenused in combination with conventional cancer therapy capable of causinggenomic damage.

In addition the use of a p53 oligo, such as OL(1)p53, will assist theinhibitory effect of other EGFR inhibitors because p53 transcriptionallyacitvates the EGFR gene (Ludes-Meyers et al., 1996; Sheikh et al.,1997).

(C) Combined Treatment of Patients with EGFR Catalytic Antibodies andOL(1)p53

Treatment schedule would include the following aspects: (1) A sample ofthe cancer will be taken to determine the mutational status of the p53gene. (2) Patients will be infused with 0.1 mg/kg/hr of the oligo forapproximately five days and will receive a bolus injection of the EGFRcatalytic antibody iv at a dose in the 1-50 mg range dependig to theturnover rate of the antibody. (3) Conventional chemotherapy will bestarted 24 hours after beginning the oligo infusion. Thechemotherapeutic agent(s) selected will be ones that do not bind toOL(1)p53 and which are capable of causing genomic damage. Suitableoligos for this use are described in U.S. Pat. No. 5,654,415, thedisclosure of which is incorporated by reference herein.

EXAMPLE II Catalytic Antibodies in Vaccination against HIV

A vaccine construct useful in the treatment of AIDS composed of a modelB cell epitope and a T helper epitope derived from gp120 is describedherein. CRAAs of the B cell epitope will be designed to elicit catalyticAbs. An exemplary B cell epitope is derived from the CD4 binding site,which is generally conserved in different HIV-1 strains. The CD4 bindingsite of gp120 is a suitable target, further, because unlike many otherepitopes, it is accessible to Abs on the native viral surface [31]. Itis known that the CD4 binding site is a conformational determinant.

In the present invention, preparation of a catalytic Ab that recognizesa specific portion of the CD4 binding site (as opposed to the entire CD4binding site) is described. Additional peptide epitopes in gp120 (orother HIV proteins) that might be suitable targets for catalytic Abswill also be identified. Because cleavage of gp120 may lead to globalchanges in the protein conformation, accompanied by loss of biologicalactivity, certain gp120 peptide epitopes may be appropriate targets ofcatalytic Abs even if they do not participate directly in HIV-1 bindingto host cells or HIV-1 interactions with intracellular components. Theseand other targets are also contemplated to be within the scope of thepresent invention.

T cell help for Ab synthesis is potentially subject to restriction indifferent individuals due to MHC polymorphism. In the present invention,mouse strains with well-defined genetic backgrounds will be used asmodels for the elicitation of catalytic immunity in response to B-Tepitope conjugates. A “universal” T-helper epitope recognizedpromiscuously by various MHC class II alleles will be utilized. Anotherbenefit of this approach is that it is readily adaptable to humanclinical trials.

The envelope glycoproteins of HIV-1 are synthesized as a single 160 kDprecursor, gp160. This protein is cleaved at the Arg511-Ala512 bond by acellular protease, producing gp120 and the integral membrane proteingp41. The biological activity of gp120 is a key ingredient in initialbinding of host cells by HIV-1, propagation of the virus, and its toxiceffects on uninfected neurons and other cells. Binding of aconformational epitope of gp120 to CD4 receptors on host cells is thefirst step in HIV-1 infection. Individual amino acids constituting thisepitope appear to be located in the second (C2), third (C3), and fourth(C4) conserved gp120 segments [12]. These are gp120 residues 256, 257,368-370, 421-427 and 457. See FIG. 7. Monoclonal antibodies that bindthe CD4 binding site have been described [32]. Since the CD4 bindingsite is a conformational epitope, distant residues that are notthemselves constituents of the epitope may be important in maintainingthe ability to bind CD4.

gp120 interactions with other host cell proteins are also essential forvirus propagation. For example, binding of gp120 by calmodulin may beinvolved in HIV-1 infectivity, as revealed by the inhibitory effect ofcalmodulin antagonists. Asp180 located between the V1 and V2 regions ofgp120 is critical for viral replication [33]. Similarly, the V3 loop maybe essential for infectivity [34]. It is clear, therefore, thatstructural determinants in gp120 other than those constituting the CD4binding site are necessary for virus genome replication, coat proteinsynthesis, and virus particle packaging.

Trypsinization of gp120 blocks its neurotoxic effects. Treatment ofHIV-1 particles with trypsin, mast cell tryptase or Factor Xa attenuatestheir infectivity. Cleavage of gp120 at residues 269-270 or 432-433destroys CD4 binding capability, whereas cleavage at residues 64-65,144-145, 166-167, 172-173 or 315-316 does not affect CD4 binding [35].On the other hand, cleavage at the Arg315-Ala316 peptide bond located inthe V3 loop of gp120 by a cellular protease is believed to be essentialfor productive viral infection. A dipeptidylpeptidase expressed on thehost cell-surface (CD26) has been proposed as being responsible forcleavage at Arg315-Ala316. This cleavage site is located in theprincipal neutralizing determinant (PND), which is a component of the V3gp120 loop to which protective Abs are readily synthesized. It has beenhypothesized that Ab binding to the PND blocks the cleavage of gp120 bya host cell protease, resulting in HIV neutralization. There is noevidence that the PND plays a direct role in HIV binding by CD4, but itsparticipation in binding by the HIV coreceptors has been suggested.

Efficient Ab synthesis by B cells is dependent in part on recruitment ofT helper cells, which, once sensitized, secrete the necessarystimulatory cytokines and activate B cells by direct contact mediatedthrough accessory molecules, such as CD4 on T helper cells and B7 on Bcells. Recruitment of Ag-specific T cells occurs through recognition bythe T cell receptor (TCR) of the complex of a processed Ag epitope boundto MHC class II molecules.

The peptide-based vaccines are formulated by covalently linking a T cellepitope to a B cell epitope, against which the host synthesizes Abs. TheT epitope binds MHC class II molecules on the surface ofantigen-presenting cells, and the MHC class II complex of the B-Tepitopes is then bound by the TCR. Different individuals in an outbredspecies express different MHC class II alleles involved in Agpresentation to T cells (I-E and I-K loci). Ideally, a peptide vaccineshould be free of MHC restrictions, i.e., a robust Ab response should beprovoked regardless of the MHC class II variations involved in Agpresentation.

The interactions between MHC class II molecules, the TCR and the Agepitope are quite promiscuous. Thus, certain peptides can serve asuniversal T epitopes, i.e., these peptides can bind the different MHCclass II alleles efficiently. Further, there is no apparent restrictionof recognition of the peptides at the level of the different types ofTCRs. Such peptides are suitable T epitope components in vaccinesdesigned to neutralize HIV through elicitation of a protective Abresponse, as described in the present invention.

As mentioned previously, certain Abs both bind and cleave peptide bondsin protein antigens. Recent studies suggest that certain germline genesencoding the V domain of L chains are capable of expressing thiscatalytic activity. Abs and L chains with comparatively nonspecificpeptidase activity (designated polyreactive activity) have beendescribed in unimmunized humans and animals [36]. Further, the catalyticresidues of a VIPase L chain identified by mutagenesis are encoded by agermline VL gene. The peptidase activity may also be improved over thecourse of somatic diversification of Abs which occurs followingimmunization with peptide antigens. Certain VIPase L chains with highlevels of catalytic efficiency have been observed to be highly mutatedin comparison to their germline gene counterparts [14]. Pairing of theappropriate VH domain with a catalytic VL domain is described to resultin improved catalytic efficiency [28]. Further, polyclonal catalytic Absisolated from patients with autoimmune disease display high affinitiesfor their autoantigens [1,4,5], which is a classical sign that the Abshave beeen subjected to somatic mutations and clonal selection.

The presence of catalytic Abs in autoimmune disease may be due to agenetic predisposition towards catalyst synthesis, brought about byselective expression of particular germline V genes or by increasedformation of catalytic sites during somatic sequence diversification ofAb V domains. The observation that autoimmune disease is associated withbiased usage of different V-genes is well-established in the literature.Other genes relevant to Ab expression may also contribute to catalyticactivity levels in autoimmune disease. The MRL/lpr mouse is known to bea good catalytic Ab producer [7]. In this mouse strain, a mutation ofthe Fas apoptosis gene is believed to permit proliferation of T and Bcells and expression of lupus-like disease.

By incorporating appropriate structure in the immunogens capable ofinducing the synthesis of Abs that combine specificity for gp120 withrapid peptide bond cleaving activity, an immunotherapeutic agent for thetreatment of AIDS will be generated.

The catalytic activity of autoantibodies to thyroglobulin and of variousL chains capable of cleaving synthetic protease substrates is inhibitedby diisopropylfluorophosphate (DFP), which reacts covalently withactivated serine residues. See FIGS. 8 and 9.

The catalytic Abs to VIP contain a high affinity antigen binding subsitethat is structurally and functionally distinct from the catalyticsubsite. In the anti-VIP L chain, mutagenesis at the residuesresponsible for chemical catalysis (Ser27a, His93) produces reductionsof turnover (k_(cat)) but minimal change in K_(m), suggesting thatresidues responsible for transition state stabilization do notcontribute in substrate ground state recognition. Mutagenesis atresidues spatially distant from the catalytic subsite produced loss ofbinding to the substrate ground state (increased K_(m)) and also a gainin turnover, as predicted. It may be concluded, therefore, that theresidues responsible for initial high affinity binding and the chemicalcleavage step are not the same.

Antibodies to Transition State Analogs (TSAs) and Covalently ReactiveAntigen Analogs (CRAAs):

Immunization with TSAs [37, 13, 38] has been proposed as a means toderive Abs that bind the transition state, and thus lower the activationenergy barrier for the reaction. As described hereinabove, the commonlyused phosphonate analogs contain a tetrahedral phosphorous atom and anegatively charged oxygen atom attached to the phosphorous. Formation ofthe transition state of peptide bond cleavage is thought to involveconversion of the trigonal carbon atom at the cleavage site to thetetrahedral state, and acquisition of a negative charge by the oxygen ofthe carbonyl group. The phosphonate TSAs may induce, therefore, thesynthesis of Abs capable of binding the oxyanion structure and thetetrahedral configuration of the transition state. However, Abs to theseTSAs, while capable of accelerating comparatively undemanding acyltransfer reactions, have not been reported to catalyze peptide bondcleavage. An antibody to a phosphinate TSA has recently been reported toslowly cleave a stable primary amide [11]. The anti-phosphinate Ab maypermit superior transfer of a proton to the amide nitrogen at thescissile bond, compared to the more common anti-phosphonate Abs, whichprobably accounts for its better catalytic activity.

In the present example, our CRAA design is predicated on the followinghypotheses: (a) as in enzymes, catalysis by Abs requires theparticipation of chemically activated amino acids to catalyze peptidebond cleavage. (For instance, the Ser hydroxyl group in serine proteasesacquires nucleophilic character and the capability to mediate covalentcatalysis due to formation of an intramolecular, hydrogen bonded networkbetween the Ser, His and Asp residues); and (b) multiple structuralelements are recognized by catalysts to achieve efficient transitionstate stabilization. It appears that the phosphonate TSA structure aloneis an incomplete immunogen for induction of catalytic Abs, because thisstructure does not contain the elements needed to bind nucleophiliccatalytic sites, or the sites in the catalysts responsible for S1flanking residue recognition site. The antigen analogs of the presentinvention induce the synthesis of Abs with covalent catalyticcapability, along with the ability to recognize basic flanking residueand the tetrahedral reaction center. Synthesis of the afore-mentionedtype of catalytic Abs induced by CRAAs designed to bind the germlineencoded, serine protease site in Abs is described herein. ElectrophilicCRAAs capable of reacting with the nucleophilic serine residue incatalytic Abs will be prepared. These novel CRAAs will be used asimmunogens, to force the utilization of the serine protease sites forthe synthesis of the gp120 specific Abs. Immunization with theaforementioned CRAAs promotes clonal selection of B cells expressing thegermline encoded catalytic sites. Further, the specificity for gp120will be ensured by incorporating an appropriate antigenic epitope ofgp120 on the flanks of the CRAA structure. See FIG. 10.

It should be noted that the conventional phosphonate TSA structure mayalso be useful, even if it is an insufficienct immunogen by itself. Theincorporation of a basic residue at the P1 site in a phosphonate TSAmight help induce catalytic Ab synthesis, because stabilization of thereaction center in the transition state can occur in conjunction withflanking residue recognition. Further, heterologous immunization, inwhich immunization with the phosphonate ester CRAA is followed byimmunization with the phosphonate TSA, might permit development of thecovalent catalytic site as well as the oxyanion stabilizing site. Absites that combine these functions will be catalytically more powerfulthan those utilizing only one of the above-mentioned mechanisms.Accordingly, methods for co-administering both TSAs and CRAAs to apatient are contemplated to be within the scope of the presentinvention.

Autoimmune disease is associated with the production of potentantigen-specific catalytic Abs. Abs capable of binding [39] and cleavinggp120 have been identified in lupus patients. Further, the L chainsisolated from lupus-prone mice (MRL/1pr strain) cleave gp120.

IgG samples purified by affinity chromatography on protein G-Sepharose[41] from 17 HIV-1 positive patients and 10 lupus patients were analyzedfor the ability to cleave ¹²⁵I-gp120. Radiolabeling ofelectrophoretically pure gp120 (IIIB, AIDS Research and ReferenceReagent Program, NIH) was by the chloramine-T method, followed bypurification of ¹²⁵I-gp120 by gel filtration. A single band ofradiolabeled gp120 at 120 kD was observed by SDS-PAGE andautoradiography. Sixteen of 17 HIV-1 positive IgG samples were devoid ofthe gp120 cleaving activity, and one showed barely detectable activity.In comparison, 3 of 10 lupus IgG samples showed readily detectable gp120cleavage. See FIG. 11. No hydrolysis of ¹²⁵I-albumin by the IgG sampleswas evident, suggesting that the observed gp120 hydrolysis is not anonspecific phenomenon. In separate experiments, L chains were purifiedfrom one of the gp120 cleaving lupus IgG samples and from the serum IgGof MRL/lpr mice. This was done by reduction and alkylation of the IgGand FPLC gel filtration, using protocols described previously forisolation of VIP cleaving L chains from human monoclonal and polyclonalIgG [9, 28]. ¹²⁵I-gp120 cleaving activity was evident in the fractionscorresponding to the L chain peak from both the the lupus patient andthe MRL/lpr mice (25 kD). The identity of the L chains recovered fromthe FPLC column was confirmed by SDS-PAGE and immunoblotting asdescribed previously [28]. Similar L chain fractions from HIV-1 positiveIgG and BALB/c IgG did not display the gp120 cleaving activity. Thespecific activities of ¹²⁵-gp120 cleavage by the lupus L chains, MRL/1prL chains and trypsin were (expressed as the reduction in the intactgp120 band area in arbitrary units/nM catalyst/h incubation time), 31,307 and 204 respectively. Note that the catalyst subpopulation probablyconstitutes a small fraction of the L chains, implying that the truespecific activity of the catalytic L chain must be greater than thevalue cited above.

In the presence of a serine protease inhibitor (0.3 mMdiisopropylfluorophosphate), gp120 cleavage by IgG from a lupus patientwas essentially completely inhibited (FIG. 12A). In comparison,inhibitors of metalloproteases, cysteine proteases and acid proteases(EDTA, iodoacetamide, Pepstatin A) were without effect on the reaction.

Further proof for L chain catalyzed gp120 cleavage has come fromidentification of a monoclonal L chain with this activity. Twenty ninemonoclonal L chains purified from patients with multiple myeloma, threerecombinant VL domains of these L chains, a recombinant L-chain with VIPhydrolyzing activity [10] and polyclonal anti-VIP Abs[2] were screenedfor the ability to hydrolyze ¹²⁵I-gp120. One monoclonal L-chain from amultiple myeloma patient with gp120 hydrolyzing activity was identified(Lay2). The remaining Ab samples were devoid of activity. The gp120hydrolyzing activity coeluted from a gel filtration column with theL-chain protein peak. Nearly equivalent cleavage of gp120 by Lay2 wasobserved in physiological buffers and nutrient media (PBS, HBSS andRPMI1640). Four radiolabeled gp120 cleavage products of massapproximately 80 kD, a smear around 50 kD, 20 kD, and <6 kD were evidentby nonreducing electrophoresis. The 80 kD band underwent furtherdiminution in size under reducing conditions, suggesting that itcontained disulfide bonded fragments. Identical product profiles wereobserved using ¹²⁵I-gp120 preparations derived from HIV-1 strains IIIB,SF2 and MN (FIG. 12B). Like the lupus IgG, the activity of the L chainwas inhibited by the serine protease inhibitor DFP, but not byinhibitors of other types of proteases.

To confirm that the cleavage reaction was not an artefact associatedwith the radioiodination of gp120, cleavage of the unlabeled protein wasstudied (FIG. 13). The cleavage products were identified byimmunoblotting of reducing SDS-electrophoresis gels with an anti-gp120antibody previously described to recognizes proteolytic breakdownproducts of the protein [35]. Increasing hydrolysis of gp120 was evidentat increasing L chain concentrations, estimated as the reduction inintensity of the 120 kD substrate band. This was accompanied byincreasing accumulation of the 80 kD and other cleavage products. Thecleavage profiles of unlabeled gp120 and radiolabeled gp120 analyzedunder reducing conditions were identical, except that the intensities ofthe individual bands were different, which is probably a reflection ofthe methods used for detection of the two types of substrates(immunoblotting versus ¹²⁵I-labeling at Tyr residues followed byautoradiography).

The initial rates of the cleavage reaction measured at 20 nM L chain andincreasing gp120 concentrations (10-300 nM) were saturable (apparentK_(m) value 30 nM; Vmax0.06 nmol gp120/nmol Lay2/h). The nM Km valuesuggests comparatively high affinity binding. Trypsin-catalyzed gp120cleavage analyzed in parallel was nonsaturable at concentrations up to 1μM, suggesting low affinity recognition. VIP inhibited the cleavage of¹²⁵I-gp120 by the L chain (K_(i) of VIP, 620 nM). The Lay2 L chain alsohydrolyzed radiolabeled VIP with a K_(m) of 144 nM [40]. Thus, VIPappears to bind the L chain about 5-21 fold less strongly than gp120.Two short regions of homology have been identified between gp120 andVIP, which might underlie reactivity of both polypeptides with thecatalyst.

Methods are provided for the synthesis of peptide analog formulationsthat elicit the synthesis of specific and efficient catalytic Abscapable of protecting against HIV infection. Earlier studies havesuggested that polyreactive catalytic activity of germline encoded Abscan be recruited and improved by immunization of mice with theserine-reactive CRAA of a gp120 peptide. The elicitation of a catalyticAb response should provide superior protection against HIV-1 infectioncompared to a noncatalytic Ab response. The following syntheticimmunogens will be prepared and assessed:

-   -   A) synthetic immunogens        -   a) the phosphonate transition state analog (TSA) of a B cell            epitope of gp120 (residues 421-436) conjugated to a T-helper            epitope from tetanus toxoid (residues 830-844) [designated            B-T epitope];        -   (b) the phosphonate ester CRAA of the B-T epitope; and        -   (c) the unmodified peptide form of the B-T epitope.    -   (B) Immunize non-autoimmune mice (strain B10.BR) and autoimmune        mice (MRL/lpr) with the three immunogens from (A) and study the        following activities of IgG purified from the sera:        -   (a) binding and cleavage of the phosphonate B-T epitope, the            phosponate ester B-T epitope and the unmodified B-T epitope;        -   (b) binding and cleavage of monomer full-length gp120; and        -   (c) binding and cleavage of native cell-surface-bound gp120.            Immunogens

The prototype vaccine capable of eliciting catalytic antibodies to HIVcontains: 1) an epitope to which B cells can make high affinityantibodies (B epitope); 2) an epitope that is bound by MHC class IIantigens and presented to T cells (T epitope); and 3) a structural mimicof the transition state formed during peptide bond cleavage, which isintended to provoke the synthesis of antibodies capable of stabilizingthe transition state, and thus catalyzing the cleavage reaction.

B Epitope component: Loss of infectivity following cleavage of gp120 canbe achieved by directing the catalyst to cleave a peptide bond locatedin an epitope of gp120that plays an important role in the infectionprocess. Note that cleavage of gp120 at a bond distant from thebiologically important determinants may also lead to loss of gp120function, because the conformation of the gp120 fragments may be altered“globally” relative to the parent protein. The probability ofneutralizing viral infectivity can be increased by directing the Ab torecognize an epitope that is a known target of neutralizing Abs.Cleavage of the CD4 binding site is an attractive mechanism to achieveHIV neutralization for the following reasons: CD4-gp120 binding is anessential step in HIV entry into host cells; cleavage of the CD4 bindingat the 432-433 bond by trypsin is known to block the ability of gp120 tobind CD4; Abs to the CD4 binding site are known to inhibit HIVinfection; the CD4 binding site on native gp120 expressed on the HIVsurface is exposed to the environment (as opposed to several otherepitopes of monomeric gp120 that are buried in native gp120 oligomers)[32]; and, the CD4 binding site is quite conserved in different subtypesof HIV-1. The linear peptide sequence composed of gp120 residues 421-436has been selected as the B epitope component of the immunogen in thepresent project (KQIINMWQEVGKAMYA; FIG. 10). Mutagenesis studies haveshown that this region of gp120 make important contributions in CD4binding.

Transition state analog component and covalently reactive antigen analogcomponent: Catalysis occurs when the transition state is stabilized morethan the ground state. In the present invention the antigen analogs actto recruit catalytic function while retaining the ability of Abs to bindthe ground state of the antigen. The latter property is necessary toobtain gp120-specific catalysts, as opposed to Abs that cleave variouspolypeptides non-specifically. Inclusion of the gp120 peptide sequencesflanking the targeted peptide bond will confer specificity for gp120.The key structural features responsible for stabilization of thetransition state of peptide bond cleavage by serine protease-likecatalytic Abs are shown in FIG. 14 and may include: (a) The tetrahedral,electrophilic carbon atom formed in the transition state at the scissilepeptide bond, capable of binding nucleophilic serine residues in thecatalyt; (b) The oxyanionic structure formed at this carbon, which canbe stabilized by ion pairing with residues like Asn, Gln or Arg in thecatalyst (the so-called oxyanion hole); and (c) The basic residue on theN-terminal side of the scissile peptide bond, recognition of which mayoccur by ion pairing with acidic residues such as Asp or Glu locatedwithin or close to the catalytic site in the Abs. Note that thepositively charged side chain of the flanking residue, although notdirectly involved in bond making and breaking processes duringcatalysis, can occupy a different spatial position in the transitionstate than in the ground state. This is possible because the partialdouble bond character of the scissile peptide bond will be lost uponformation of the transition state, permitting rotation around this bond,and consequent changes in the positions of remote groups. Thefeasibility of such remote spatial changes in the transition state hasbeen deduced by computational modeling of a peptide substrate in the sp2(ground state) and sp3 (transition state) configurations at the scissilebonds.

A TSA and a CRAA which comprise phosphonate analog and a phosphonateester analog, respectively, will be assessed. In both cases, thetetrahedral phosphorous atom serves as the analog of the scissilepeptide bond carbon atom linking residues 432 and 433 in gp120. In thephenylester configuration shown in FIG. 10, the phosphorous atomacquires a partial positive charge, just as the scissile bond carbonatom carries the partial positive charge required for its reaction withnucleophilic serine residues. Peptidic O-phenylphosphonates havepreviously been described to be capable of irreversibly inactivatingvarious serine proteases by forming a covalent bond with the oxygen atomof the active site serine residue [29]. Sampson and Bartlett and others[23, 24] have established the chemical synthesis needed to prepare thephenyl ester at the phosphorous atom, and to attach peptide sequencesflanking the phosphonate ester.

Twelve and four amino acids are present, respectively, on the N and Cterminal sides of the TSA/CRAA structure, corresponding to the sequenceof residues 421-436 of gp120. A basic residue has been incorporated atthe P1 position of the CRAA-gp120 to exploit the existence of thegermline encoded, basic residue-specific catalytic site in Abs. Thepresence of the basic residue, along with the phosphonate phenylesterstructure, promotes tight binding to catalytic site, and thus promotesthe ability of the CRAA-gp120 to selectively stimulate the clonalproliferation of B cells synthesizing the catalytic sites.

It should be noted that the above phosphonate ester CRAA of the Bepitope is structurally distinct from previous phosphonate TSAs appliedto raise esterase Abs [13, 38]. The conventional phosphonate TSAscontain an anionic oxygen attached to the phosphorous, which can bindthe oxyanion hole found in the catalysts. The phosphonate TSAs, however,can not react with nucleophilic serine residues in the catalytic site. Aphosphonate TSAs of the Phe-Ile peptide bond reportedly did not inducethe formation of amidase catalytic Ab formation [41].

The phosphonate ester analog described above will be compared to aphosphonate TSA of the B epitope for the following reasons: (a) theimmunogen described in the afore-mentioned study did not contain a basicresidue at the P1 position, which would work against recruitment of thegermline catalysts for synthesis of peptidase Abs; and (b) whileimmunization with a phosphonate analog alone may be insufficient toprovoke peptidase Ab synthesis, heterologous immunization with thephosphonate and phosphonate ester analogs may lead to a good peptidaseAb response, because the heterologous immunization can be anticipated toselect for the oxyanion hole (phosphonate immunization) as well as thenuqleophilic serine residues (phosphonate ester immunization). Such acoimmunization using the gp120 phosphonate and phosphonate esterimmunogens is contemplated to be within the scope of the presentinvention.

T epitope component: To recruit T cell help for synthesis of anti-gp120Abs, a fifteen amino acid peptide (QYIKANSKFIGITEL) corresponding toresidues 830-844 of tetanus toxin will be placed on the N terminal sideof the B epitope. The presence of the T epitope in the vaccine constructeliminates the need to conjugate the B epitope to a large carrierprotein. Several previous studies have shown that comparatively shortlinear peptides that include a T and a B epitope are capable ofprovoking efficient Ab synthesis to the B epitope [42]. The tetanustoxin T epitope to be employed in the present invention is known toserve as a T epitope in hosts expressing diverse class II alleles, andhas been characterized, therefore, as a “universal” T epitope [43].Further, a gp120 B epitope linked to this T epitope is described toinduce anti-gp120 Ab synthesis. The “universality” of the T epitope,although deduced from human studies, probably extends to the mouse,because class II restrictions tend to be conserved phylogenetically.Regardless of the possible differences on this point between man andmouse, the mouse strains to be utilized in the present invention havebeen matched for class II alleles involved in recruitment of T cell helpfor Ab synthesis (A^(k)E^(k) haplotype), eliminating concern thatdifferential T helper recruitment might contribute to variations incatalytic Ab responses.

Assembly of immunogens: Synthesis of the 31 residue ground state B-Tconstruct (designated unmodified B-T epitope) composed of tetanus toxinresidues 830-844 at the N terminus and gp120 residues 421-436 at the Cterminus will be done by conventional solid phase synthesis on anApplied Biosystems synthesizer. Mass spectrometry and ¹H and ¹³CNMR willbe done to confirm the structures.

The TSA and CRAA of the B-T epitope will contain the phosphonate and thephosphonate ester structures at the targeted cleavage site. These arenovel reagents, but their synthesis should not present problems.Standard organic chemistry techniques utilized previously for synthesisof TSAs and other types of enzyme inhibitors [23,24].

A brief overview of the synthetic scheme is as follows. The phosphinateisostere of lysine will be prepared from the diphenylmethlyamine salt ofhypophosporus acid and 6-benzylcarbamatohexanal, followed by removal ofthe diphenylmethyl group in acid. The required flanking peptides(tetanus toxoid residues 830-844 extended with gp120 residues 421-431;gp120 residues 432-436) are prepared by conventional solid phasesynthesis, except that the peptide corresponding to the C terminalfragment contains 2-hydroxy-6-carbobenzyloxyaminohexanoic acid insteadof the N terminal lysine. Other basic side chains are protected with thecarbobenzyloxy group and acidic side chains are protected with a benzylester group. Protected peptides will be attached to the phosphinatelysine isostere by classical solution phase peptide synthesis methods.The final peptide phosphonate phenyl ester structure will be prepared byoxidative coupling of the phosphinate with phenol. This same synthesisscheme will be used used for preparation of the phosphonic acid byconverting the phosphinate to the phosphonic acid monoester by treatmentwith bis(trimethylsilyl)acetamide in acetonitrile followed by aqueoustriethylamine, carbon tetrachloride, and lithium exchange on AG-X-50 ionexchange resin [23; scheme h and I]. Mass spectrometry and NMR will bedone to confirm the structures.

Immunization of Mice

Two strains of mice will be studied for Ab responses to 4 immunogenconstructs, BR10.BR and MRL/lpr. Immunizations will be done with:

-   a. B-T epitope (residues 421-436 of gp120 linked to residues 830-844    of tetanus toxin).-   b. Phosphonate analog of the B-T epitope at residue Lys432. (TSA)-   c. Phosphonate ester analog of the B-T epitope at residue Lys432.    (CRAA)-   d. Phosphonate ester analog followed by phosphonate analog the B-T    epitope (TSA+CRAA; heterologous immunization).

Conventional immunization methods will applied to induce Ab synthesis.Three intraperitoneal and one intravenous injection of the immunogens(about 100 μg peptide each) will be administered. The final immunizationwill be carried out intravenously. Two adjuvants will be tested: RIBIand alum. Alum is approved for human use and has previously been shownto provoke Ab synthesis to a B-T epitope similar to those proposed inthe present invention. RIBI is a low toxicity replacement for Freund'sComplete Adjuvant, and reproducibly facilitates good Ab responses to avariety of Ags. Sera will be prepared from retroorbital plexus bleedsobtained from the mice at five time points over the course of theimmunization schedule. Splenocytes will be harvested and processed forpreparation of Fv phage display libraries for structure-functionstudies. Analysis of two adjuvants is advantageous because the qualityand magnitude of Ab responses to vaccines can be influenced byadjuvants, via effects of the cytokines and TH subpopulations recruitedby the adjuvants on B cell development and clonal selection [44]. Thus,a total of 16 groups of mice will be studied (4 immunogens×2 mousestrains×2 adjuvants), each composed of 5 animals.

Low affinity, antigen-nonspecific peptidase antibodies are alreadypresent in preimmune repertoire. Provided that the germline geneencoding the nonspecific peptidase activity is recruited for the Absynthesis, immunization with the CRAAs will result in synthesis ofgp120-specific catalytic Abs. Humans and mice with autoimmune diseaseare prolific producers of Ag-specific catalytic Abs, suggesting that thediseased immune system efficiently recruits the germline gene encodingthe catalytic site, and permits maturation of the catalytic sites tobecome specific for individual Ags over the course of the immuneresponse. The MRL/lpr mouse strain is genetically prone to autoimmunedisease, and has previously been observed to be capable of high levelcatalytic antibody production. Further, the L chains from the serum ofpreimmune MRL mice express gp120 cleaving activity. Thus, a subset ofantibodies formed by immunization of MRL/lpr mice with the disclosedimmunogens can be anticipated to express gp120-specific catalyticactivity. It is relevant that gp120-binding Abs found in lupus patientsare directed, in part, to the gp120 B epitope included in the disclosedimmunogens [39]. The BR10.BR mouse strain is not prone to autoimmunedisease. The results from this strain will reflect the ability of thedisclosed immunogens to stimulate catalytic immunity to gp120 in thehealthy immune system. B10.BR mice and MRL/lpr mice have identicalhaplotypes at the class II loci responsible for T cell restriction of Absynthesis (A^(k)E^(k)), ensuring that any differences in catalytic Absynthesis between the two strains will not be due to the class IIrestriction.

Ab Binding Activity

Abs synthesized in response to the CRAAs should bind the transitionstate of the peptide bond cleavage reaction better than the groundstate, permitting catalysis to occur. The strength of the binding of theAbs to the CRAAs/TSAs will serve as an predictor of the Ab catalyticactivity. Further, if the B epitope in the immunogen exists inapproximately the same conformation in the immunogen and the full-lengthgp120, the Abs will also bind the full-length protein. Finally, if theepitope is exposed as in the native gp120 known to exist in the form ofoligomers on the viral surface, the Abs should also bind the oligomericgp120 structure.

Unmodified B-T epitope and TSA B-T epitope and CRAA B-T epitope: Thebinding of the three forms of the B-T epitope, i.e., the unmodified,phosphonate, and the phosphonate ester form, will be compared by ELISA.Apparent values of binding strength will be assessed by competitionassays (as IC50 values), using ELISA plates coated with the unmodifiedB-T epitope (about 50 μg/ml) as the solid phase and the unmodified,phosphonate and phosphonate ester B-T epitope as the soluble competitor.The binding will be measured using peroxidase coupled anti-mouse IgGfollowed by addition of the substrates (o-phenylene diamine and hydrogenperoxide). Since polyclonal preparations are to be studied, the bindingcurves may deviate from simple sigmoidal binding isotherms. The IC50values for the individual ligands will serve, nevertheless, as validindicators of the average binding affinity of the Abs.

The following relationship could be applied to predict the catalyticrate acceleration: Ki/Kd=k_(cat)/k_(uncat), where Ki and Kd are theequilibrium dissociation constants of the TSA and the unmodified B-Tepitope, respectively, and k_(cat) and k_(uncat) are the first orderrate constants for the catalyzed and uncatalyzed reactions,respectively. IC50 values could be substituted in this equation topredict the rate acceleration, but the predicted value will be anaverage of the activity of several Abs, because the IgG samples to bestudied are mixtures of different Abs. The binding assays will beconducted at 4° C. in the presence of diisopropylfluorophosphate (DFP)to minimize interference with measurement of the binding parameters dueto peptide cleavage. DFP, a known serine protease inhibitor, has beenobserved in previous studies to uniformly inhibit the catalytic activityof Abs. Reduction of the reaction temperature will reduce the rate ofthe catalytic reaction. Note that Km values estimated from catalysisassays will help confirm the validity of the IC50 as an indicator ofbinding strength.

Solution phase assays will be conducted to confirm that avidity effectsdue to the antigen “carpeting” on the solid phase do not lead tomisleading binding estimates. The solution phase assays will be carriedout using the ¹²⁵I-radiolabeled B-T epitope. Radiolabeling of thepeptide will be done using the chloramine-T method as describedpreviously [2]. [The B-T epitope contains one Tyr, corresponding togp120 residue 435]. Ab-Ag complexes will be trapped using proteinG-Sepharose and the binding determined by counting the radioactivity ina γ-spectrometer. As before, the binding will be studied at variousconcentrations of the TSA/CRAA competitors, permitting estimation of thebinding strength of the unmodified epitope and its TSA/CRAA.

Purified gp120 and cell-surface expressed gp120: Ab binding by purifiedgp120 and cell-surface gp120 will be measured to determine whether thetargeted B epitope is accessible to the Abs in the full-lengtholigomeric form of the protein. Recombinant gp120 expressed in amammalian cell line will be employed to assure that the glycosylationpattern of the protein is similar to that in HIV-infected cells.Competitive ELISA using gp120 coated on a solid phase will be performedto determine the apparent binding strengths of the Abs. Competitorligands to be studied are the full-length gp120 and the three B-Tepitopes to which the Abs are elicited (phosphonate TSA, phosphonateester CRAA and the unmodified B-T epitope). The relative reactivity ofthe synthetic immunogens and the full-length gp120 will be estimatedfrom the IC50 values (apparent Ki) of the competitor ligands. Nearequivalent IC50 values for the full-length gp120and the unmodified B-Tepitope will indicate that the targeted B epitope exists in anear-equivalent conformation ln the two molecules. Observationsindicating stronger binding of the Abs to the phosphonate or thephosphonate ester of the B-T epitope compared to full-length gp120 willindicate that the Abs may display catalytic activity. As describedabove, solution phase assays using radiolabeled gp120 will be carriedout to confirm the absence of ELISA artefacts, such as increased bindingavidity due to the ligand immobilization.

HIV-1 infected cells of the H9 T cell line express gp120 on theirsurface. The majority of cell-surface gp120 mimics the form of adherentvirus particles. The cell-surface gp120 is thought to exist in anoligomeric state similar to the aggregation status of the gp120 on thesurface of the virions. Ab reactions with the cell-surface expressedgp120, thus, have been held to indicate the ability of the Abs torecognize virion-bound gp120.

In the present invention, H9 cells obtained from the NIH AIDS Repositorywill be grown in RPMI/10% FCS in 5% CO₂. The cells (10⁶/ml) are infectedwith the culture supernatant containing HIV-1 strain MN (AIDSRepository) for 2 hours at 37° C. Following washing, the cells arecultured for about 1 week. Binding of various concentrations of IgG (1nM-1 μM) will be determined by incubation with an appropriate number ofthe intact cells for 4-6 h at 4° C. in round-bottomed 96 well plates inthe presence of 1 mM DFP, followed by washing of the cells to removeunbound Ab, incubation with rabbit antimouse IgG conjugated toperoxidase, development of the reaction with hydrogen peroxide ando-phenylenediamine, and quantitation of the optical density at 490 nmusing an ELISA reader. Controls will include the preimmune IgG and an Abknown to be reactive with cell-surface gp120 (available from the NIHAIDS repository). Ab binding to the cells can also be studied by flowcytometry, using a fluorescent second Ab for detection of the boundanti-gp120 Ab as described [45]. This procedure permits determination ofapparent Ab affinity by estimation of Ab association and dissociationrates.

Competition experiments will be carried out in which the B-T epitopeconstructs or soluble full-length gp120 will be permitted to act ascompeting ligands for Ab binding to the cells. As in the competitionstudies described in the preceding paragraph, the IC50 values of the B-Tepitopes will estimate the relative strengths of the Ab binding to the Bepitope. To minimize gp120 cleavage by the Abs, the incubations will beconducted at 4° C. Diisopropylfluorophosphate, which is an effectiveinhibitor of Ab catalysis, can also be included in the incubations. Cellviability will be estimated at the end of the binding reaction by trypanblue exclusion tests, to confirm that the inhibitor (and otherexperimental conditions) does not disrupt cellular integrity, whichcould potentially perturb the oligomeric structure of the gp120.

Screening for Catalytic Activity:

IgG purified from sera by affinity chromatography on protein G-Sepharosewill be screened for catalytic activity using the unmodified B-T epitopeas the substrate. Initial screening assays will be carried out at 10 μMsubstrate and 0.25 μM IgG concentrations with incubations times of 1-2hours. Even with an apparent turnover as low as 0.1/min, the productsshould accumulate to concentrations of about 3 μM (30% of initialsubstrate concentration). The reaction mixtures will be analyzed byreversed-phase HPLC with detection at 214 nm (trifluoraceticacid/acetronitrile gradient). Product concentrations will be computedfrom areas under the product peaks. Controls will includepreimmunization IgG.

All IgG samples will also be screened for cleavage of full-length gp120.¹²⁵I-gp120 will be the substrate. Radiolableing of gp120 (recombinant MNexpressed in CHO cells) is by the chloramine-T method followed byresolutive FPLC to obtain electrophoretically homogeneous ¹²⁵I-gp120.SDS-electrophoresis and autoradiography will be applied to visualizeproduct bands. Procedures permitting rapid sample handling have beendescribed. About 50 IgG samples can be screened for the activity perday. Unlabeled gp120 will be used as substrate to confirm that thecleavage reaction is not an artefact associated with the radiolabelingprocedure. Immunoblotting of SDS-PAGE gels of the reaction mixtures withan anti-gp120 Ab capable of recognizing various proteolytic fragments ofgp120 will be applied for this purpose.

Purity of the IgG used as catalyst will be established by SDS-PAGE.Retention of the catalytic activity in the Fab fractions and IgGprepared by gel filtration under denaturing conditions (6 M guanidiniumchloride) will confirm that the catalytic activity is due to Abs aspreviously described [36].

The assays will be done in the absence and presence of human serum toassess whether protease inhibitors found in serum influence catalytic Abactivity. In preliminary studies, serum has been found to be withouteffect on gp120 or thyroglobulin cleavage by Abs isolated from lupusserum. Based on these results, serum inhibitor-resistant Abs should alsobe present in mice.

Cleavage site specificity: The product fragments generated by cleavageof the unmodified B-T epitope and of full-length gp120 will beidentified, permitting deduction of the cleavage site(s). The B-Tepitope fragments separated by HPLC will be subjected to N-terminalamino acid sequencing and FAB-mass spectrometry to identify the cleavagesite(s), as previously described (1,2). In the case of the full-lengthgp120 substrate, the reaction products will be separated bySDS-electrophoresis, blotted onto PVDF and the blotted polypeptidessubjected to N-terminal sequencing, permitting identification of thecleavage sites by comparison with the sequence of gp120. Controls willinclude IgG from preimmune mice.

Trypsin will be included as a positive control. Trypsin can be expectedto cleave the B-T epitope at multiple peptide bonds that are flanked bya Lys or Arg residue, i.e., at residues 421-422 and 432-433 in the Bepitope and at residues 833-834 and 837-838 in the T epitope. Incomparison, recruitment of the catalytic activity in Abs due to thepresence of the phosphonate or phenylphosphonate ester structure, shouldcleave the the B-T epitope mainly at the Lys432-Ala433 peptide bond byIgG. It is possible that an alternative result may be observed.Ag-specific catalysts can be synthesized by immunization with groundstate antigens. Thus, catalysts capable of cleaving the substrates atpeptide bonds other the 432-433 bond may be found, because the germlineencoded activity present in the preimmune repertoire may recognize basicresidues without regard to the overall structure of the antigen epitope[36, 40]. The extent to which the cleavage reaction occurspreferentially at residues 432-433 will indicate the importance of thephosphonate/phenylphosphonate ester structure in recruiting thecatalytic site. Similarly, the extent to which the cleavage offull-length gp120 is confined to peptide bonds located within residues421-436 will indicate the importance of this peptide epitope inrecruiting catalytic activity that is specific for gp120.

Substrate specificity These studies will be performed to assess thetherapeutic use of the antibody catalysts. Cleavage of various peptideand protein substrates will be studied. Several radiolabeledpolypeptides are available to study the substrate-specificity profile:(a) ¹²⁵I-albumin; (b) ¹²⁵I-thyroglobulin; (C) ¹²⁵I-VIP; and, (d)¹²⁵I-IgG. Hydrolysis of the proteins is indicated by appearance of lowermass product bands visualized by electrophoresis and autoradiography.VIP hydrolysis is measured by precipitation of the intact peptide withtrichloroacetic acid or by reversed-phase HPLC [2]. A larger panel ofrandomly selected polypeptides (n>10; commercially availablepolypeptides, e.g., casein, collagen, etc.) will also be examined byinhibition assays, i.e., their ability to inhibit 125I-gp120 hydrolysisby the catalyst. Inhibition of the reaction is indicated by reduceddepletion of the 120 kD gp120 band.

Kinetics: Kinetic studies will be done to determine the apparent rateconstant and catalytic efficiency (k_(cat)/K_(m)). The unmodified B-Tepitope and full-length gp120 will be used as substrates. The kineticconstants will be determined from assays conducted at varyingconcentrations of the substrates. If the the sera contain catalytic Absmixed with noncatalytic Abs capable of binding the epitope, the bindersmay protect the epitope from cleavage by the catalyzers. Todifferentiate between these two pools, the serum IgG will be adsorbedonto an immobilized inhibitor that can binds the catalytic Abs but notthe noncatalytic Abs. Such inhibitors are available. Because suchinhibitors do not contain a gp120 epitope, they will not bindnoncatalytic Abs induced by immunization with the gp120 B-T epitope.Biotin will be attached to the inhibitor to permit immobilization usingavidin coated plates. Following incubation of the serum IgG from immunemice with excess immobilized inhibitor, the supernatant will be analyzedby ELISA for binding to the unmodified B-T epitope. An absence ofbinding will suggest that the noncatalytic Abs to the epitope are notpresent in significant amounts. Further, the bound Abs may be elutablewith hydroxylamine, pH 9 or greater, which may cleave the covalent bondbetween the Abs and the inhibitor. The eluted Abs, which will bedeficient in noncatalytic Abs to the B-T epitope, can then be analyzedfor kinetic parameters.

Nanomolar or lower Km values will indicate high affinity antigen bindingactivity. Observations of equivalent Km and kcat values for the twosubstrates will suggest that residues 421-436 located in the syntheticimmunogen adopt a conformation similar to that in gp120. Concerningvalues of the apparent rate constant, the IgG preparations shoulddisplay more rapid turnover than that observed previously usingpolyclonal Ab preparations, because the immunogens of the invention areexpressly designed to promote the recruitment and improvement ofcatalytic sites over the course of the immunization procedure.

The inhibition of IgG catalyzed cleavage of the unmodified B-T epitopeby the phosphonate and phosphonate ester of the epitope will also beassessed. Reduced hydrolysis will indicate that the phosphonate andphosphonate ester peptides are competitive inhibitors of the binding ofthe B-T epitope and/or serve as alternate substrates. When inhibition isseen, the K_(i) value will be measured to assess the relative reactivityof the IgG with the B-T epitope and its TSA/CRAAs. To determine whetherthe TSA/CRAAs are used as substrates, their cleavage by the IgG will bestudied by RP-HPLC separation of product peptides and identification ofthe product peptides by amino acid sequencing. If the catalysts arecapable of cleaving multiple peptide bonds promiscuously, the TSA/CRAApeptides may be cleaved by the catalysts. On the other hand, if the Abscleave exclusively at the Lys432-Ala433 bond, the TSA/CRAAs will notserve as substrates because they contain noncleavable analogs of thepeptide bond at this position.

Cleavage of cell-surface expressed gp120: To confirm that the catalyticantibody activity is directed to the biologically relevant form of thegp120, i.e., virion bound oligomeric gp120. Biosyntheticallyradiolabeled gp120 expressed on the cell surface will serve as thesubstrate. gp120 radiolabeling will be done by growing HIV-infected H9cells in ³⁵S-labeled methionine (in Met-deficient medium). The cellswill be treated with varying concentrations of the IgG fractions frompreimmune and immune mice. Cell extracts will be prepared using a milddetergent (0.1% Triton-X-100), which should be sufficient to release theradiolabeled gp120 into the supernatant. The gp120 (and its fragments)will be immunoprecipitated from the cell extracts using an availablerabbit anti-gp120 Ab, which is known to bind various tryptic fragmentsof the protein [35]. SDS-electrophoresis will be employed to separatethe reaction products. Disappearance of the intact gp120 band andappearance of lower mass fragments will indicate the cleavage ofcell-surface bound gp120. Controls will include immunoprecipitation ofthe cell extracts with nonimmune rabbit IgG, in which case noradioactivity should be precipitated. Immunoblotting of the gels withanti-gp120 Abs will be carried out to confirm that theimmunoprecipitated material represents gp120 fragments.

Confirmatory experiments that the Abs recognize the conformation ofgp120 expressed on the surface of HIV-1 will also be performed. Sucrosedensity gradient purified MN-virus preparations as the substrate(available from Advanced Biotechnologies) will be used. Followingincubation of the virus with the Abs, gp120 cleavage will be determinedas described for cell-surface expressed gp120, i.e., detergentextraction, electrophoresis and immunoblotting with the anti-gp120 Abknown to bind gp120 cleavage fragments.

Immunization with the B-T epitope immunogens will elicit Abs that bindfull-length soluble and cell-surface expressed gp120, because thetargeted epitope in gp120 is a part of the CD4 binding site, which canbe assumed to be exposed on the protein surface (as opposed to beingburied in the interior of the protein). Moreover, the targeted epitopeis conserved in different HIV strains. Thus, synthesis of broadlyreactive Abs is expected.

Preimmune IgG from non-autoimmune mice will be devoid of the ability tocatalyze the cleavage of the targeted epitope in gp120. Similarly, IgGfrom non-autoimmune mice immunized with the unmodified B-T epitope willexpress little or no gp120 cleaving activity.

Preimmune IgG from autoimmune mice may display low-level cleavage ofgp120, but the activity will not be highly specific for gp120.Immunization of the autoimmune mice with the unmodified B-T epitope willrender the catalytic activity specific for gp120, but improvements incatalytic turnover are not predicted from the structure of theimmunogen. In comparison, the B-T epitope TSA and CRAA are designed toprovoke the synthesis of Abs that combine the ability to bind the groundstate of gp120 as well as the transition state of the peptide bondcleavage reaction. Thus, the TSA and CRAA immunizations are predicted toelicit the synthesis of Abs that display bind gp120 with high affinity(low values of apparent Km and Kd) and display rapid turnover (apparentkcat). Further, immunization of the autoimmune mice with the analogs ofthe B-T epitope will direct the promiscuous catalytic activity found inthe preimmune state to one specialized to recognize the targeted gp120epitope (residues 421-436).

Immunization of B10.BR mice (non-autoimmune mice) with the TSAs andCRAAs will overwhelm the suppressor mechanisms that limit catalytic Absynthesis in the non-autoimmune state. This test is relevant todevelopment of an HIV vaccine, because the goal is to develop vaccinesthat protect against the infection, regardless of the autoimmune ornon-autoimmune status of the host.

The phosphonate ester of the B-T epitope will elicit more potentcatalysts than the phosphonate B-T epitope, because the former immunogenwill promote clonal expansion of B cells synthesizing Abs containingnucleophilic Ser/Thr residues, which is a feature of the pre-existingcatalytic sites encoded by germline VL gene(s). The phosphonate B-Tepitope, on the other hand, is designed to recruit Abs that contain anoxyanion hole (such as Asn255 in subtilisin) to stabilize the developingnegative charge on the carbonyl oxygen in the transition state. Noevidence is available that proves that oxyanion stabilization isresponsible for catalysis by the germline encoded catalysts. Note,however, that V region somatic diversification mechanisms(hypermutation, V-J/V-D-J recombination and VL/VH pairing diversity) arepowerful mechanisms capable of evolving catalytic sites de novo.Development of Abs that combine the germline nucleophilic site and asomatically developed oxyanion hole is quite feasible. Suchnucleophilic, oxyanion stabilizing sites are responsible for efficientcatalysis by non-Ab serine proteases. The proposed heterologousimmunizations, in which Ab synthesis will be induced by sequentialimmunization with the phosphonate ester B-T epitope and phosphonate B-Tepitope will provoke the synthesis of high turnover, gp120-specificcatalysts. The heterologous immunization will also recruit the Abgermline gene(s) encoding nucleophilic sites due to the covalent,electrophilic reactivity of the phosphonate ester, followed by somaticdevelopment of an oxyanion stabilizing structure over the course of theimmune response.

Comparison of HIV-1 Neutralizing Activity of Anti-gp120 AntibodiesElicited by the Unmodified B-T Epitope with the Neutralizing Activitiesof Abs Elicited by the Phosphonate B-T Epitope TSA and the PhosphonateEster B-T Epitope CRAA.

Binding of gp120 to CD4 initiates infection of cells by HIV-1. Cleavageof gp120 in at the 432-433 bond will efficiently block HIV-1 binding bycells, because the cleavage site is located in the CD4 binding region ofgp120, and cleavage of this bond by trypsin has previously been shown toinhibit solution phase gp120 binding by CD4 [35].

Abs: Purified IgG samples from mice at various time over the course ofthe immunization with the control B-T epitope construct (unmodifiedpeptide), the phosphonate B-T epitope (TSA), the phosphonate ester B-Tepitope (CRAA), and the combination of the phosphonate and phosphonateester B-T epitope (TSA+CRAA) will be compared. Hyperimmune IgG from allof these immunizations should be capable of high affinity gp120 binding.The catalytic activity is anticipated to be present in the IgG from theTSA/CRAA immunizations from both mouse strains.

HIV-1 neutralization: Initially, blinded IgG samples from all of themice at various stages of immunization will be screened in awell-characterized, quantitative, T-cell line assay using a standardlaboratory strain (HIV-1 MN). Further studies will be done usinghyperimmune IgG obtained towards the end of the immunization schedule.Controls will include cells incubated with IgG without HIV-1 to rule outthe possibility of a nonspecific toxic effect of the IgG. These IgGsamples will be analyzed using blood-derived PHA-activated lymphocytesas one cell type and blood-derived macrophages as the second cell type.A single dual-tropic primary isolate HIV-1 ADA will be the virus isolateused in the primary cell assay. The reason for using both cell types isto avoid missing any neutralizing/inactivating activity which may residein a unique epitope specific to the different cell and virus types. Itis apparent from earlier work that a number of factors are responsiblefor the profound differences in the neutralization of laboratory strainsfrom field isolates. Those Ab samples exhibiting neutralizing activitywill be directly compared for their potency to both V3 andCD4-inhibiting human monoclonals and well characterized HIV-1 positivehuman polyclonal sera. In addition, these antibodies will be furtherevaluated for their stage of, mechanism(s) of action, reversibility aswell as their breadth of neutralizing activity over a wide range ofantigenic subtypes/clades in multiple primary cell types.

The IgG to be tested will include the noncatalytic anti-gp120 (fromnon-autoimmune mice immunized with the unmodified B-T epitope) andcatalytic anti-gp120 IgG preparations (e.g., from mice immunized withthe TSA/CRAAs of the B-T epitope). Because the targeted B epitope ingp120 is essential for CD4 binding, even noncatalytic Abs in IgGpreparations of the invention can be anticipated to inhibit HIV-1neutralization. Assuming that sufficient titers of the Abs are elicited,hyperimmune IgG from each of the experimental groups of mice may inhibitthe HIV-1 infectivity.

Homogeneous preparations of catalytic and one noncatalytic Fv constructswill be compared for HIV-1 neutralization activity. This will confirmthe results obtained from the polyclonal IgG studies. Further, becausethe Fv constructs lack the Fc domain, phenomena like complement bindingand Fc receptor binding will be eliminated. The absence of enhancedHIV-1 infectivity due to such phenomena will be thus be confirmed usingthe Fv constructs.

The major attraction of catalytic Abs is their greater and irreversibleantigen neutralizing capability compared to noncatalytic Abs. In thepresent invention, the catalytic IgG samples should display potent HIV-1neutralization, at concentrations several orders of magnitude lower thanthe noncatalytic IgG samples. The epitope targeted by the catalyst is aconstituent of the CD4 binding site of gp120. Further, cleavage of thetargeted bond (Lys432-Ala433) by trypsin has been found to block gp120binding to CD4. The CD4 binding site tends to be conserved acrossdifferent strain and subtypes of HIV-1. Thus, the anti-gp120 catalystsof the present invention represent a beneficial therapeutic tool for thetreatment of infectious disorders, such as HIV infection.

EXAMPLE III Use of CRAAs and Catalytic Antibodies inIschemia-Reperfusion Injury and Septic Shock/SIRS

Ischemia-reperfusion injury occurs when blood supply to a tissue isinterrupted for a prolonged period (ischemia) and then restored(reperfusion). This type of injury affects both heart attack and strokepatients following treatment to restore blood flow to the damagedtissue. Both ischemia and particularly reperfusion are associated withrelease into this tissue of certain factors that cause an inflammatoryresponse and injury by inducing programmed cell death.

Septic shock and systemic inflammatory response syndrome (SIRS) areterms for a frequently fatal syndrome that includes hemodynamic changes,inflammation and ultimately the failure of major organs in a predictableorder beginning with the lungs. The septic shock syndrome was originallyassociated only with gram-negative bacterial infections and the effectsof endotoxin, but subsequently a variety of other medical problems, suchas extensive tissue damage resulting from an accident, were found toinitiate the same syndrome, which in the absence of infection is termedSIRS [46]. The multiple organ failure seen in both syndromes is closelyassociated with and may largely be caused by the occurrence ofprogrammed cell death.

Ischmia-Reperfusion Injury

The four major soluble factors that induce programmed cell death in thisdisorder are reactive oxygen species (ROS) and nitric oxide(→peroxynitrite→hydroxyl ROS) which induce and are induced byinterleukin-1 beta (IL-1) and tumor necrosis factor alpha (TNF).

Considering the involvement of programmed cell death inischemia-reperfusion injury and septic shock/SIRS, the fact that thesoluble factors just mentioned play a prominent role in both underscoresthe similarities in pathophysiology between the medical emergencies.

The novel CRAAs of the invention may be used to advantage to developcatalytic antibodies which cleave IL-1 and TNF for the treatment ofischemia-reperfusion injury, septic shock/SIRS and acute respiratorydistress syndrome (ARDS) as well as for other inflammatory disorderssuch as rheumatoid arthritis and for the treatment of neuropathic pain.

Ischemia-Reperfusion Injury

Early return of blood flow to ischemic tissues is critical in haltingthe progression of cellular injury that results from an interruptedoxygen and nutrient supply. Paradoxically, the reinstitution of bloodflow to ischemic tissues is associated with further tissue damage. Ithas been shown experimentally, for example, that four hours ofintestinal ischemia is substantially less damaging than three hours ofischemia plus one hour of reperfusion [47, 48]. The importance of thereperfusion phase to overall tissue damage has been illustrated innumerous studies showing that therapeutic interventions initiated duringthe ischemic phase are only as effective as those initiated at the onsetof reperfusion [49, 50, 51, 52]. Ischemia-reperfusion injured tissuesrapidly show zones of necrotic cell death surrounded by areas of cellsundergoing programmed cell death [53, 54, 55].

It is well established that ischemic tissues must be exposed tomolecular oxygen upon reperfusion to exhibit injury [56-61].

Several mechanisms have been postulated to explain the pathogenesis ofischemia-reperfusion injury but most attention has focused on ROS. ROSrefers to any compound derived from molecular oxygen that has a negativecharge including superoxide, hydrogen peroxide and the hydroxyl radicalwhich are reduced by one, two and three electrons respectively.

Numerous lines of evidence have implicated ROS in ischemia-reperfusioninjury including the following: 1) The production of ROS in ischemictissues has been detected by electronic spin resonance and spin trapping[62, 63] as well as by nitroblue tetrazolium reduction,chemiluminescence, and salicylate trapping. 2) Exposure of tissues toROS in the absence of ischemia-reperfusion injury produces pathologicchanges similar to ischimia-reperfusion injury itself [64, 65, 66, 67].3) Treatment with agents that scavange ROS or limit ROS productionsignificantly reduce ischemia-reperfusion injury damage [68, 69].

One of the initial effects of ischemia is ATP depletion in the affectedtissue, which in turn makes cell membranes permeable to ions, andcalcium sequestration inefficent. The resultant increase in cytosoliccalcium promotes activation of calcium-dependent phospholipase andproteolytic enzymes, and an important result is the conversion ofxanthine dehydrogenase into xanthine oxidase [70]. Xanthine oxidase isfound in parenchymal and endothelial cells, and produces ROS superoxideand, directly or indirectly, hydrogen peroxide. That the inhibition ofxanthine oxidase reduces the damage caused by ischemia-reperfusioninjury supports the notion that ROS production by this enzymecontributes to pathogenesis.

Reduction in phosphatidylethanolamine, breakdown of phospholipids, andliberation of free fatty acids occurs in ischemic tissues. With theonset of reperfusion there is rapid utilization of free fatty acids,particularly arachidonic acid, which stimulates the lipoxygenase andcyclo-oxygenase pathways resulting in the production of ROS.Cyclo-oxygenase inhibitors have been shown to be beneficial in reducingtissue damage due to ischemia-reperfusion injury.

Nitric oxide is a highly reactive species continually released by theendothelium [71]. It maintains the microcirculation in a state of activevasodilation and vascular impermeability and prevents platelet andleukocyte adherence to the endothelium. It is enzymatically synthesizedby a consitutively active endothelial synthase from L-arginine and itsproduction can be inhibited by L-arginine analogues such asN³-nitro-L-argine methyl ester (L-NAME). Inhibition of nitric oxideproduction in the coronary vasculature with inhibitors such as L-NAMEcan cause myocardial ischemia by vasoconstriction [72]. There is asubstantial body of evidence, however, that nitric oxidesynthesisinhibitors can substantially reduce the level of tissue damageassociated with ischemia-reperfusion injury [73, 74].

Inducible forms of nitric oxide synthase are responsible for increasedlevels of this molecule during ischemia-reperfusion injury. Inducersinclude inflammatory cytokines, neuroexcitatory amino acids, andflow-related vasodilation during postischemic hyperthermia. Noiri et al.[75] demonstrated that an antisense oligo targeting transcripts ofinducible nitric oxide synthase genes can reduce the expression of thesegenes. Given systemically, this oligo was taken up by the kidney andsignificantly reduced renal failure caused by the experimentalproduction of renal ischemia in rats.

Nitric oxide can combine with the superoxide anion to produce the toxicfree-radical peroxynitrite, leading to the production of the hydroxylradical, a ROS thought to be a major causal factor inischemia-reperfusion injury [74]. Reduction of molecular oxygen toproduce superoxide occurs in all aerobically respiring cells in themitochondria transport system.

Nitric oxide has also been shown to induce programmed cell death in anumber of physiologic and experimental situations. Activation ofhigh-level nitric oxide production helps form the first line of defenceagainst invading pathogens and tumor cells.

Release of ROS in areas of ischemia-reperfusion injury attractsinflammatory leukocytes, which in turn can cause tissue injury by meansof a cytotoxic arsenal that includes the release of additional ROS.Numerous studies have shown: 1) that leukocytes accumulate in areas ofischemia-reperfusion injury, and 2) that depletion of circulatingneutrophils or use of agents that prevent neutrophil activation cansometimes reduce tissue damage associated with ischemia-reperfusioninjury.

The terminal phases of programmed cell death involve a set of enzymesbelonging to the ICE family. Peptides capable of inhibiting some ofthese enzymes have been shown to reduce ischemic brain damage resultingfrom transient middle cerebral artery occlusion in rodents andsignificantly improve resulting behavioral deficits [76]. The latterobservation demonstrates that functional recovery of ischemic neuraltissue can follow treatments that prevent the cell death program fromgoing on to completion. Presumably, the degree of functional recoverywould be even greater in instances where ischemia is followed byreperfusion injury.

Numerous studies have demonstrated that programmed cell death is aubiquitous feature of tissue damaged by ischemia-reperfusion injury [53,54, 55].

Induction of inducible nitric oxide synthase levels has been positivelycorrelated with programmed cell death in rat hearts by Szabolcs et al.[77]. Cardiac tissue was transplanted from Lewis to Wistar-Furth rats asa model of cardiac allograft rejection, while Lewis to Lewis transplantsserved as a control. The number of cardiac myocytes undergoingprogrammed cell death increased sharply from day 3 to day 5 followingtransplantation. At day 5, allografts showed a significantly greaterincrease in the myocytes, endothelium and macrophages undergoingprogrammed cell death when compared to syngenic grafts. Expression ofinducible nitric oxide synthase mRNA, protein and enzymatic activity wasshown to increase in parallel in time and extent with programmed celldeath in the cardiac myocytes. Immunohistochemical staining demonstratedthat areas of increased inducible nitric oxide also expressednitrotyrosine, indicative of peroxynitrite formation.

Numerous lines of evidence support the conclusion that interleukin-1beta (IL-1) and tumor necrosis factor alpha (TNF) play important rolesin the evolution of ischemia-reperfusion injury.

The production of both proinflammatory cytokines has been shown to beassociated with cell activation of the monocyte/macrophage series, whichcan occur as the result of xanthine-oxidase-derived oxygen radicalactivity.

IL-1 was discovered in the 1940s and was initially shown to producefever when injected into animals. In the early 1970s, IL-1 was found tohave a variety of other biological effects when injected into animalsincluding neutrophilia, heightened antimicrobial responses, increasedsynthesis of hepatic acute phase reactants, and induction of colonystimulating factors. It was also found to boost T-cell response tomitogens in culture and to function as an adjuvant.

Cloning studies have demonstrated that IL-1 is a three-member familyconsisting of IL-1 alpha, IL-1 beta and IL-1ra. The first two areagonists and the last a receptor antagonist. IL-1 alpha is localized oncell membranes while IL-1 beta is released as a cytokine and is the formsimply referred to in this text. It is synthesized as a precursor thatmust be cleaved before it can become active. The most specific of theseenzymes is interleukin-lbeta-converting enzyme (ICE) which is closelyrelated to the family of enzymes active in the final phases ofprogrammed cell death.

IL-1 expression has been demonstrated in areas subjected toischemia-reperfusion injury in the retina, liver, skeletal muscle andintestine. Both IL-1 and TNF expression were similarly demonstrated inthe brain and heart. In the rodent model used by Hara et al. [76], IL-1expression reached its peak 30-60 minutes after reversal of experimentalocclusion of the middle cerebral artery and decreased thereafter.

Treatment of rat cardiac myocytes with IL-1 induces nitric oxidesynthase transcription and increased expression of the enzyme by aprotein kinase A-dependent pathway. IL-1 was also shown to induce thisenzyme in brain endothelial cells. Similarly IL-1 and TNF were shown toinduce heart and hepatic nitric oxide synthase. As discussed above, ROScause genomic damage that induces p53 expression which can result inprogrammed cell death.

IL-1 also induces the expression of other proinflammatory cytokines suchas IL-6. In some instances induction is mediated by the transcriptionfactor NF-KB which can also mediate the effects of TNF. The frequentlyobserved synergy between IL-1 and TNF as well as IL-1 and IL-6 may beexplained in part on the basis of significantly overlapping signaltransduction pathways in cell populations responsive to all threecytokines.

IL-1ra treatment of rats undergoing hepatic ischemia-reperfusion injuryhas been found to reduce TNF production, tissue injury and mortality[78]. Ischemia was induced in rat livers by clamping the vessels of theleft and middle lobes for 90 minutes. In one set of experiments, IL-1rawas given systemically five minutes before ischemia was induced, and TNFlevels were determined in the blood and liver at various time pointsafter reperfusion had begun. In control animals, TNF levels in bothtissues were found to increase over time as the reperfusion continued,with the experiment being terminated 4½ hours from the initiation ofischemia. In contrast, IL-1ra treatment caused a decrease in TNF levelsin the two tissues. Histologic examination demonstrated that IL-1ratreatment was associated with substantially less liver damage comparedto controls.

In a second set of experiments, the unaffected right lateral and caudatelobes of the liver were removed after the period of ischemia wascompleted. Eighty percent of control animals died compared to 30% ofthose treated with IL-1ra.

In similar studies, it was demonstrated that IL-1ra treatment andnaturally occuring IL-1ra protect rat brain tissue fromischemia-reperfusion injury-induced damage.

The role of TNF in ischemia-reperfusion injury of the brain wasexamined. In one set of experiments, variable doses of TNF wereadministered intracerebroventricularly to rats 24 hours before occludingthe middle cerebral artery for 80 or 160 minutes (transient) or untiltermination of the experiment 24 hours later (permanent). In some groupsTNF neutralizing antibody was given 30 minutes before the TNF injection.Administration of exogenous TNF produced a significant dose-dependentincrease (32%) in the infact size caused by permanent occlusion. Thehigh dose of TNF (25 pmol) caused an increase in the infact size in bothtransient occlusion groups of 100% and 34% respectively. All of theseeffects of exogenous TNF were abrogated in the animal that receivedpretreatment with the TNF antibody.

In yet another set of experiments, the effects of blocking endogenousTNF was evaluated by blocking TNF function with either a neutralizingantibody or a soluble TNF receptor (sTNF-RI) given 30 minutes before or3 or 6 hours after permanent occlusion. Blocking TNF function before orafter occlusion resulted in an up to 26% reduction in infact sizedepending on the inhibitor dose.

TNF has been shown to partially mediate liver damage associated with thereperfusion phase of ischemia-reperfusion injury. Hepatic TNF productionwas responsible for neutrophil sequestration and activation in theaffected area, leading to the release of ROS. Passive immunization withneutralizing TNF antibodies could significantly inhibit these pathogeniceffects. It has also been shown that TNF administered to culturedhepatocytes enhances the cytotoxicity of ROS given at the same time.Similarly, neutralizing TNF antibodies were shown to reducecardiovascular effects and improve survival rate after acuteischemia-reperfusion injury was induced by a 45-minute occlusion of thesuperior mesenteric artery in a rat model.

Septic Shock/SIRS

Leaving etiology aside, the basic pathophysiologic events that occur inischemia-reperfusion injury and septic shock/SIRS are very similar, butin the former the pathology is localized while in the latter it issystemic and can terminate in multiple organ failure beginning with thelungs. Key elements in both groups of disorders are ROS, nitric oxide,IL-1, TNF, and programmed cell death.

Most of the experimental studies in this field involve septic shockbecause of the ease with which the syndrome can be induced usingbacteria or bacterial products. The pathophysiologic changes uncoveredin these studies associated with septic shock/SIRS in patientsdemonstrate that the syndrome is driven by a cascade of proinflammatorymediators. It is generally agreed that this cascade is initiated by IL-1and TNF which are initially released from macrophages and otherinflammatory cells. IL-1 is also produced by a wide variety of othercell types and most of the cells in the body have receptors for IL-1.IL-1 production has been shown to be stimulated by ROS and TNF and bothof these cytokines promote ROS production. In septic shock, IL-1 and TNFproduction results from the action of endotoxin and other bacteriaproducts. The high expression of these two factors along with ROS leadsto the excessive production of a wide variety of secondary mediatorsincluding IL-6, IL-8, gamma-interferon, prostaglandin I₂, thromboxaneA₂, prostaglandin E₂, transforming growth factor beta, plateletactivating factor, bradykinin, angiotensin, and vasoactive intestinalpeptide. These factors contribute to the pathological cardiovascular,hemodynamic and coagulation and other changes associated with thissyndrome.

TNF was the first cytokine to be linked to the septic shock/SIRsyndrome, when it was demonstrated that its overproduction is anantecedent to shock and death. Soon after IL-1 was shown to be similarlytoxic and was synergistic when given with TNF. As a result otherwisenonlethal amounts of TNF and IL-1 when combined, produced lethal shockin animals.

Following an inflammatory insult, TNF is the first cytokine to appear inthe circulation followed by IL-1. In volunteers injected with endotoxin,for example, TNF levels peak 60-90 minutes after the insult and returnto baseline within three hours. IL-1 levels plateau 3-4 hours aftertreatment. These general observations and the finding that TNF caninduce IL-1 production have contributed to the notion that TNF is theinitial cytokine that begins the septic shock/SIR syndrome whereas IL-1is more involved with its continuation.

Measurement of serum IL-6 levels, which is induced by both TNF and IL-1,has been suggested as a better measure of TNF and IL-1 production than adirect measurement of these cytokines. IL-6 levels in the circulationoften directly correlate with the severity of disease in patients withtrauma, sepsis and septic shock/SIRS. Unlike IL-1 and TNF, however, IL-6administration does not induce inflammation or septic shock/SIRS andinhibitors of IL-6 do not prevent the lethal effects of this syndrome.

A large body of additional evidence supports the notion these mediatorsplay a critical role in the pathogenesis of this syndrome. Casey et al.[77], for example, examined the correlation between the various factorsinvolved in septic shock and the outcome for 97 patients, 57 of whicheither had full blown septic shock or were hypotensive which is an earlyindicator of impending septic shock/SIRS. The survival rate for thisgroup of patients was 54%. The strongest positive correlation wasbetween plasma IL-6 levels and mortality and the second strongest waswith the IL-1 levels. IL-1 is usually undetectable in normal subjects(<40 pg/ml). There was no correlation between TNF, and endotoxin levelsand death. The lack of correlation with TNF was ascribed to the factthis cytokine is only produced in the earliest stages of the factorcascade and it has a short half-life. Elevated TNF levels, however, didcorrelate with the presence of gram positive sepsis.

The proinflammatory cytokines involved in septic shock/SIRS, particularyTNF, activate the coagulation and complement cascades which causesneutrophil activation with the release of ROS. Nitric oxide synthase isinduced by IL-1 and TNF in both endothelial cells and inflammatorycells. Activated neutrophils consume oxygen in the so called respiratoryburst forming the super oxide ion that reacts with nitric oxide to fromperoxynitrite that decomposes to from the hightly toxic hydroxyl ROS.ROS, especially superoxide, generate chemotactic factors when they reactwith a plasma precursor in a self-amplifying process.

Patients with septic shock/SIRS have responded favorably to treatmentwith anti-oxidants confirming the importance of ROS in the pathogenesisof this syndrome. A marked reduction in mortality rate, for example, hasbeen seen in patents with the sepsis-related acute respiratory distresssyndrome (ARDS) following treatment with a four anti-oxidant combinationtreatment. ARDS refers to the lung-failure related pathophysiology thatis the first of the multiple organ failures that characterisze theterminal phase of septic shock/SIRS. Similarly, in another studypatients with ARDS given the antioxidant n-acetyl cysteine showedimproved lung and cardiac function including changes in pulmonaryvascular resistance, cardiac output and oxygen delivery.

The mechanistic understanding of septic shock/SIRS that has developedbased on these and a large body of additional data strongly suggeststhat this syndrome could be prevented by agents that block TNF and IL-1production. IL-1 neutralizing antibodies have been shown to amelioratethe septic shock syndrome in animal models. Recombinant IL-1raadministration, however, has been the most frequently used approach forblocking IL-1 function in animal models of septic shock/SIRS [78].

Simultaneous treatment of rabbits, for example, with adequate amounts ofIL-1ra followed by normally lethal quantities of endotoxin result inonly mild and transient hypotension and decreased neutrophilinfiltration into tissues. IL-1ra has also been demonstrated to preventthe death of rats infected with K. pneumoniae and from E. coliperitonitis.

The ability of IL-1ra infusion to attenuate subsequent lethal E. coliseptic shock in baboons has been studied. When given in excess in therange of 10³ to 10⁴ fold with respect to IL-1 levels, IL-1ra prevented asustained IL-1 response, although no effect on the initial production ofthe cytokine was seen, resulting in a 100% survival rate at 24 hours vs.43% in placebo treated controls.

Many reports have provided evidence that TNF neutralizing antibodies andsoluble TNF receptors can protect animals from otherwise lethalinjections of bacterial toxins, such as endotoxin, that can induceseptic shock. To be beneficial, however, these treatments have to begiven before or during the infusion of endotoxin or bacteria.

The role of TNF in the initiation of septic shock has also beeninvestigated using TNF-1 receptor knockout mice. These mice were shownto not respond to doses of TNF that produces a lethal septic shocksyndrome in normal mice. The knockouts also did not respond to normallylethal doses of endoxin if they were pretreated with D-galactosamine andagent that sensitizes animals to the toxin by blocking its metabolism bythe liver. There were no differences between the normal mice and theknockouts in terms of the plasma levels of TNF induced by endotoxin.Following a sublethal challenge with endotoxin, the levels of IL-6released in to the circulation were found to be dramatically less in theknockout mice compared to controls. Finally, macrophages from knockoutmice have been shown to be severely limited in their capacity to producenitric oxide by the inducible nitric oxide synthase pathway. Incontrast, mice with an IL-6 deletion showed no such deficit.

As many as six TNF antagonists under development by five differentcompanies for the treatment of septic shock have been in Phase II-IIIclinical trials at one time. Four of these inhibitors were neutralizingantibodies and two were soluble recombinant TNF receptors. None of theseproducts has has distinguished itself as a viable treatment for thissyndrome. TNF antagonists including neutralizing antibodies, however,are not intrinsically without efficacy in patients because they havesubsequently shown substantial promise in clinical trials for thetreatment of rheumtoid arthritis.

Clearly, attempts over the last ten years to develop new treatments forseptic shock/SIRS have resulted in many disappointments. Pruitt et al.[78] have summarized a number of the reasons put forth by numerousinvestigators for why the cytokine inhibitor trials have failed. Perhapsthe single greatest obstacle to success relates to the cost of theexisting cytokine inhibitors. Their high price precludes them from beingused prophylactically. For example, as pointed out by Pruitt et al.[78], to sustain a therapeutic plasma concentration of 10-15micrograms/ml, IL-1ra has to be given at concentrations of 1.5-2.0mg/kg/hr of about 2.5 grams per day for as long as the patient isseptic.

Consequently, patients who receive IL-1 or TNF inhibitors are alreadysymptomatic. Yet our understanding of the pathogenesis of this syndromestrongly suggests that IL-1 and particularly TNF function must beblocked at the very initiation of the cascade of excessiveproinflammatory cytokine release that has a major role in driving thesyndrome forward. Again, animal studies have convincingly shown that tobe effective IL-1 and TNF must be given prior to or simultaneously withthe inflammatory insult that engenders the septic shock/SIR syndrome.

In the Synergen Inc. Phase II trial, for example, IL-1ra was given 9hours on average after a patient was judged a suitable candidate forstudy [79]. As a result, numerous patients entered into the trial haddeveloped a septic response 24 or more hours before the initiation ofthe IL-1ra infusion. Thus for many patients the proinflammatory cascadeis well advanced by the time the attempt to block IL-1 function wasbegun.

Positive responses in the IL-1ra clinical trials may have also beenobscured by the use of the 28 day all-cause mortality criterion. Are-evaluation of the Phase II and first Phase III trial data revealsthat the major benefits obtained from the IL-1ra infusion occurred 3-7days after treatment [78]. Survival curves were subsequently essentiallyparallel. This finding could reflect the very short half-life of IL-1ra.The beta-phase half-life of IL-1ra in septic primates, for example, isapproximately 21 minutes. Similarly, the half-lives of IL-1 and TNF aremeasured in minutes to hours. It is not surprising, therefore, thatmortality after the first week following IL-1ra infusion is irrelevantto assessing the value of the therapy.

Yet another problem with the IL-1ra clinical trials, which is generallyapplicable to systemically administered inhibitors of IL-1 or TNF, comesfrom the practice of determining the dose of the cytokine inhibitorbased on the plasma concentration of the cytokine. The reason is thatlocal tissue concentration of these cytokines can be much higher thanwhat is present in plasma. In patients with ARDS, for example, IL-1concentrations in the lungs have been shown to be as high as 15 ng/mlwhile the plasma concentrations are under 100 pg/ml.

Thus the septic shock/SIRS data and current understanding of thesyndrome supports the development of novel therapies which can (1) blockIL-1 and TNF and be sufficiently inexpensive to be givenprophylactically to at risk patients; and (2) prevent the programmedcell death that is a major contributing factor to the organ failure thatcauses the many deaths associated with this syndrome.

Accordingly, CRAAs are described herein which will stimulate the immuneproduction of catalytic antibodies specfic for TNF and IL-1. Theseantibodies may be administered using protocols already developed forimmunotherapies based on the administration of other known monoclonalantibodies. As the antibodies of the present invention actcatalytically, the dosage will be much lower than antibodies with bindreversibly and stoichiometrically. Accordingly, the cost of prophylactictreatment for patients at risk for the syndrome will be greatly reduced.An exemplary CRAA for eliciting catalytic antibodies to TNFA is shown inFIG. 15. Exemplary CRAAs for eliciting catalytic antibodies to IL1β areshown in FIGS. 16 and 17. A boronate electrophillic center is shown inFIG. 17.

EXAMPLE IV Passive Immunization with the Catalytic Antibodies of thePresent Invention

There are many areas in medicine where monoclonal antibodyadministration is providing clinical benefit. In the field of organtransplantation, a MoAb (OKT3) which binds to the T cell receptor hasbeen employed to deplete T cells in vivo. Additionally, MoAbs are beingused to treate graft v. host disease with some success. A clinical trialhas been established which is assessing the ability of anti-CD4 moAB todeplete a subset of T cells in the treatment of multiple schlerosis.

Accordingly, methods of administration of monoclonal antibodies are wellknown to clinicians of ordinary skill in the art. An exemplary methodand dosage schedule are provided in a phase III, randomized, controlledstudy of chemotherapy alone or in combination with a recombinant moAB tothe oncogene HER2.

All patients randomized to the recombinant humanized MoAb Her2 arm ofthe study will receive treatment as a 4 mg/kg I.V. loading dose on Day 0(the first day of the MoAb HER2 infusion, or the day of randomizationfor patients in the control group), then weekly as a dose of 2 mg/kgI.V. through out the course of the study. All patients will be monitoredduring each study visit by a clinical assessment, a symptom directedphysical examination (if appropriate) and laboratory tests. Routinetumor evaluations will be conducted for all patients at prescribedintervals during the study. All adverse events will be recorded.

The administration of the catalytic antibodies of the present inventionwill be done as described above for the HER2 monoclonal antibody. As inthe HER2 study, following infusion, patients will be assessed todetermine the efficacy of the administered catalytic antibody.

Should the catalytic antibodies administered as above give rise toundesirable side effects in the patient, the immunizing CRAAs will beadministered to covalently inhibit the action of the catalyticantibodies.

EXAMPLE V Active Immunization Using the CRAAS of the Present Invention

Active immunization will be done using previously developed methods withvaccines designed to elicit protective antibody responses against thedesired antigens [82, 83]. For example, the CRAAs mixed with a suitableadjuvant formulation such as alum can be admimistered intramuscularly ata dose optimized for maximum antibody synthesis (100-1000 μg/kg bodyweight), and two or three booster injectijns can be administed at 4 weekintervals, until the catalytic antibody concentration in the serumreaches plateau levels. The protective immunity so generated isanticipated to last for several years, because vaccination will resultin formation of specific, long lived memory cells that can be stimulatedto produce antibodies upon exposure to the offending organism or cancercell. Descriptions and methods to determine the catalytic antibodyconcentrations are set forth in Examples I and II. Because antibodysynthetic response to most antigens are T cell dependent, an appropriateT cell epitope can be incorporated into the immunogen by peptidesynthesis, as described in the case of the gp120, Example II.Alternatively, a carrier such as keyhole limpet hemocyanin can beconjugated to the CRAA via coupling through lys side chain amino groupsor Cys side chain sulfahydryl groups to maximize the antibody responseif necessary.

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method for stimulating production of antibodies with catalyticactivity comprising: a) administering to a test subject, an immunogenicamount of a covalently reactive antigen analog (CRAA); b) repeating stepa) as necessary to ensure effective antibody production; and c)isolating and purifying said antibodies; wherein said covalentlyreactive antigen analog contains an electrophilic center flanked bypeptide residues derived from proteins associated with a peptide antigento be targeted for cleavage.
 2. A method of stimulating production ofcatalytic antibodies as claimed in claim 1, wherein an immunogenicamount of a transition state analog (TSA) is co-administered with saidCRAA.
 3. A method for passively immunizing a patient, comprising: a)administering to said patient a catalytic antibody specific for anantigen associated with a medical disorder diagnosed in said patient,said catalytic antibody being produced by the method of claim 1; b)repeating step a) as necessary to maintain immunity; and c) assessingsaid patient's sera for the presence of catalytic antibodies.
 4. Amethod for actively immunizing a patient, against a microbial infection,comprising: a) complexing a covalently reactive antigen analog (CRAA)comprising an immunogenic microbial epitope from an infectious organismwith an adjuvant, said CRAA-epitope-adjuvant complex comprising avaccine; b) administering said vaccine to said patient in a dose in therange of 100-1000 micrograms/kg body weight; c) administering at leastone booster injection, said at least one booster injections beingadministered at four week intervals; and d) assessing said patient'ssera for the presence of catalytic antibodies against said microbialepitope.